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DNA interrogation by the CRISPR rna-guided endonuclease cas9
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.
... Once guide-target complementarity is validated (57,58), the nuclease domains of Cas9 generate double-stranded breaks (DSBs) in the target DNA. Although mismatched spacer sequences can be tolerated, strict PAM requirements often limit Cas9 targeting (58,59). ...
... Once guide-target complementarity is validated (57,58), the nuclease domains of Cas9 generate double-stranded breaks (DSBs) in the target DNA. Although mismatched spacer sequences can be tolerated, strict PAM requirements often limit Cas9 targeting (58,59). For instance, the density of PAM sequences from the most widely used Cas9, SpyCas9 from Streptococcus pyogenes, is only ∼1 PAM (5 ′ -NGG-3 ′ ) every 10 bases in the regions upstream of endogenous promoters in the E. coli genome (50.5% GC) (25). ...
... However, PAM expansion can lead to extended timescales for target recognition and increased off-target effects (58,59,109). To address off-target effects, both rational engineering and enzyme selection approaches have been used to develop high-fidelity SpyCas9 variants with reduced off-target activity (110)(111)(112)(113)(114)(115). ...
In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.
... The sgRNA-Cas complex searches for a suitable target site in a stochastic manner via threedimensional diffusion, sampling several target sites for complementarity [79,80]. We propose that tightly packed regions could overload the CRISPR machinery with a large number of noncomplementary target sites, forcing the sampling of more sites and lowering the probability of encountering a suitable one (Fig 6A). ...
... We propose that tightly packed regions could overload the CRISPR machinery with a large number of noncomplementary target sites, forcing the sampling of more sites and lowering the probability of encountering a suitable one (Fig 6A). The initial association between the CRISPR complex and DNA occurs through PAM (protospacer-adjacent motif) recognition [79,81,82]; despite the speediness of dissociation from a non-suitable site [80], a dense genomic region would provide an abundance of potential PAMs (e.g. almost any "GG" dinucleotide, in the case of SpCas9), significantly slowing the process. ...
CRISPR is a gene editing technology which enables precise in-vivo genome editing; but its potential is hampered by its relatively low specificity and sensitivity. Improving CRISPR’s on-target and off-target effects requires a better understanding of its mechanism and determinants. Here we demonstrate, for the first time, the chromosomal 3D spatial structure’s association with CRISPR’s cleavage efficiency, and its predictive capabilities. We used high-resolution Hi-C data to estimate the 3D distance between different regions in the human genome and utilized these spatial properties to generate 3D-based features, characterizing each region’s density. We evaluated these features based on empirical, in-vivo CRISPR efficiency data and compared them to 425 features used in state-of-the-art models. The 3D features ranked in the top 13% of the features, and significantly improved the predictive power of LASSO and xgboost models trained with these features. The features indicated that sites with lower spatial density demonstrated higher efficiency. Understanding how CRISPR is affected by the 3D DNA structure provides insight into CRISPR’s mechanism in general and improves our ability to correctly predict CRISPR’s cleavage as well as design sgRNAs for therapeutic and scientific use.
... The CRISPR/Cas9 system consists of the Cas9 nuclease, CRISPR RNA (crRNA), and transactivating crRNA (tracr-RNA) [16,17]. The Cas9 nuclease recognizes the protospacer adjacent motif (PAM), which is located downstream of the 3' end of the protospacer on the nontarget DNA strand [18]. Subsequently, the crRNA and tracrRNA combine to form a sgRNA, which guides the Cas9 nuclease to achieve DNA cleavage through complementary base pairing. ...
... In addition to selecting base editors with low off-target activity, the following points should be noted when selecting base editors for HSC gene editing: 1. PAM restriction. Most BEs currently use the SpCas9 nuclease, which recognizes the NGG PAM [18]. However, this may limit the editing scope of base editors. ...
Base editors, developed from the CRISPR/Cas system, consist of components such as deaminase and Cas variants. Since their emergence in 2016, the precision, efficiency, and safety of base editors have been gradually optimized. The feasibility of using base editors in gene therapy has been demonstrated in several disease models. Compared with the CRISPR/Cas system, base editors have shown great potential in hematopoietic stem cells (HSCs) and HSC-based gene therapy, because they do not generate double-stranded breaks (DSBs) while achieving the precise realization of single-base substitutions. This precise editing mechanism allows for the permanent correction of genetic defects directly at their source within HSCs, thus promising a lasting therapeutic effect. Recent advances in base editors are expected to significantly increase the number of clinical trials for HSC-based gene therapies. In this review, we summarize the development and recent progress of DNA base editors, discuss their applications in HSC gene therapy, and highlight the prospects and challenges of future clinical stem cell therapies.
Graphical Abstract
... However, PAM recognition is conserved among Cas9 orthologs, which trigger directional target DNA unwinding, R-loop formation and expansion, which eventually lead to reorientation of the HNH endonuclease domain to DNA cutting and concomitant RuvC activation leading to concerted DNA cleavage 27 . Recent mechanistic studies showed that the directional PAM-duplex DNA unwinding serves as the rate-limiting checkpoint of Cas9 action and a conformational switch discriminates Cas9 DNA binding and cleavage events 21,[28][29][30][31][32] . Moreover, the loss of nucleobasespecific interaction between the target DNA and Cas9 was reported to be rescued by base non-specific Cas9 interactions 3,33 . ...
The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.
... Intriguingly, expression of G1-Cas9 showed a significant effect in enhancing SSA, suggesting that SSA-mediated DSB repair operates in G1 as well as in the S-G2 phase ( Fig. 3f and Supplementary Fig. 9f). One might think that G1 occurrence of SSA needs to be interpreted with caution because Cas9-induced DSBs are unique in that they are RNAmediated; in addition, G1-Cas9 protein could possibly persist at the break site until the S-G2 phase to cause SSA [41][42][43] . In this respect, it is important to mention that G1-Cas9 expression is highly inefficient in enhancing HR ( Fig. 3e and Supplementary Fig. 9e). ...
Homology-dependent targeted DNA integration, generally referred to as gene targeting, provides a powerful tool for precise genome modification; however, its fundamental mechanisms remain poorly understood in human cells. Here we reveal a noncanonical gene targeting mechanism that does not rely on the homologous recombination (HR) protein Rad51. This mechanism is suppressed by Rad52 inhibition, suggesting the involvement of single-strand annealing (SSA). The SSA-mediated gene targeting becomes prominent when DSB repair by HR or end-joining pathways is defective and does not require isogenic DNA, permitting 5% sequence divergence. Intriguingly, loss of Msh2, loss of BLM, and induction of a target-site DNA break all significantly and synergistically enhance SSA-mediated targeted integration. Most notably, SSA-mediated integration is cell cycle-independent, occurring in the G1 phase as well. Our findings provide unequivocal evidence for Rad51-independent targeted integration and unveil multiple mechanisms to regulate SSA-mediated targeted as well as random integration.
... In addition, they showed that the Cas9 protein could work with dual tracrRNA, and crRNA-derived single guide RNA sequences programmed to cleave specific sites in the target DNA, thus providing an alternative simple method for gene targeting and gene editing [1]. In a subsequent study published in January 2014, Doudna and colleagues showed the integral role of the trinucleotide protospacer adjacent (PAM) motif in the binding of Cas9 to target DNA and the precise cleavage of DNA [35]. ...
Clustered regularly interspaced short palindromic repeats (CRISPR) based gene-editing has begun to transform the treatment landscape of genetic diseases. The history of the discovery of CRISPR/CRISPR-associated (Cas) proteins/single guide RNA (sgRNA)-based gene-editing since the first report of repetitive sequences of unknown significance in 1987 is fascinating, instructive, and inspiring for future advances. The recent approval of CRISPR-Cas9-based gene therapy to treat patients with severe sickle cell anemia and transfusion-dependent beta thalassemia has renewed hope for treating other hematologic diseases, including patients with germline predisposition to hematologic malignancies, who would benefit greatly from the development of CRISPR-based gene therapies. The purpose of this manuscript is three-fold: first, a chronological description of the history of CRISPR-Cas9-sgRNA-based gene editing; second, a brief description of the current state of clinical research in hematologic diseases, including selected applications in treating hematologic diseases with CRISPR-based gene therapy; and third, the current progress in gene therapies in inherited hematologic diseases and bone marrow failure syndromes, to hopefully stimulate efforts towards developing these therapies for patients with inherited bone marrow failure syndromes and other inherited conditions with germline predisposition to hematologic malignancies.
... As nt G16, A17 and G21 are embedded within BH helix and REC1 domain, nt C18-C20 are exposed to the bulk solvent (Fig. 1e). This suggests that nt C18-C20 might serve as the nucleation site and play a key role in the binding of TS-DNA, as proposed in previous reports 20,32 . ...
CRISPR-Cas9 has been developed as a powerful gene editing tool, but the mechanism governing the intricate catalytic process remains incompletely resolved. Here, the cryo-electron microscopy structures of thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) in complex with sgRNA and target DNA are reported. The structure of GeoCas9 in complex with sgRNA reveals a slit termed L1-crevice comprising HNH, RuvC, and L1 helix as a transient storage site of 5’ spacer of sgRNA. When 5’ spacer is extracted to pair with the target DNA, L1-crevice collapses to trigger the subsequent HNH domain translocation. In addition, structural and biochemical analyses suggest that the resilience of GeoCas9 at elevated temperature is related to the unique PI domain conformation. These results advance our understanding into the catalytic process of Cas9 and unveil the molecular mechanism that accounts for the superior thermal profile of GeoCas9.
... De manera general, los PAM son secuencias conservadas de ADN (de 2 a 6 pares de bases) que forman parte del genoma de los virus y de otros MGEs, pero no de las bacterias(Shah et al. 2013). En el sistema CRISPR/Cas de Streptococcus pyogenes, por ejemplo, la Cas9 se une al PAM trinucleótido 5'-NGG-3' para posteriormente "interrogar" a la secuencia de ADN adyacente a través del ARN guía en busca de complementariedad(Sternberg et al. 2014). Si las bases del ARN guía y la hebra de ADN interrogada son complementarias, la Cas9 cataliza un DSB tres bases río arriba de la secuencia PAM a través de dos dominios conservados con actividad nucleasa: los dominios HNH y RuvC, los cuales escinden tanto la hebra de ADN blanco (cuyas bases son complementarias a la secuencia guía del sgRNA) como la hebra opuesta a esta, respectivamente(Jiang y Doudna, 2017).2.3.3 ...
Due to environmental constraints and the continuous evolution of agricultural pests and diseases, we need new crop protection strategies to supply sufficient, nutritious, and safe food for an ever-growing global population. In plants, CRISPRa-based transcriptional regulation and epigenetic editing tools offer versatile and reversible strategies for disease resistance enhancement compatible with these goals. However, despite its potential in agriculture, the application of the CRISPRa system has been restricted to model plants with few traits of agronomic interest. An alternative to conventional model plants is tomato (Solanum lycopersicum L.). In particular, the Micro-Tom tomato variety exhibits genetic and horticultural traits, making it an attractive model for studying the CRISPRa system in vivo. Here, we report the integration of the CRISPRa system for transcriptional activation and epigenetic reprogramming of the PR-1 gene in Micro-Tom tomato plants. We achieved the generation of edited plants by stably transforming hypocotyl explants with Agrobacterium. These plants were resistant to hygromycin and developed from organogenic and embryonic structures. Additionally, we implemented a transient transformation method in cotyledonary explants as an alternative to the agroinfiltration method without adverse effects associated with Agrobacterium inoculation. This work represents an advancement in implementing the CRISPRa system for disease resistance improvement in plants of agronomic importance.
... In 2023, the structure of SFV in complex with its receptor VLDLR (PDB:81HP) was found, and avian MXRA8 (PDB:8EWF) was found to be able to bind with SINV/WEEV. replacement of specific nucleic acid sequences (Cong et al., 2013;Mali et al., 2013;Sternberg et al., 2014). Published in 2018 by Zhang et al., an article discovered that MXRA8 could play a crucial role in the entry of several alphaviruses in both humans and mice ( Figure 8). ...
Background
Alphaviruses are a diverse group of pathogens that have garnered considerable attention due to their impact on human health. By investigating alphavirus receptors, researchers can elucidate viral entry mechanisms and gain important clues for the prevention and treatment of viral diseases. This study presents an in-depth analysis of the research progress made in the field of alphavirus receptors through bibliometric analysis.
Methods
This study encompasses various aspects, including historical development, annual publication trends, author and cited-author analysis, institutional affiliations, global distribution of research contributions, reference analysis with strongest citation bursts, keyword analysis, and a detailed exploration of recent discoveries in alphavirus receptor research.
Results
The results of this bibliometric analysis highlight key milestones in alphavirus receptor research, demonstrating the progression of knowledge in this field over time. Additionally, the analysis reveals current research hotspots and identifies emerging frontiers, which can guide future investigations and inspire novel therapeutic strategies.
Conclusion
This study provides an overview of the state of the art in alphavirus receptor research, consolidating the existing knowledge and paving the way for further advancements. By shedding light on the significant developments and emerging areas of interest, this study serves as a valuable resource for researchers, clinicians, and policymakers engaged in combating alphavirus infections and improving public health.
... Once integrated into the genome, CRISPRs are transcribed, and the virus-derived sequences form short guide RNAs that are bound by the DNA endonuclease CRISPR associated protein 9 (Cas9). Consequently, binary complexes formed by guide RNA-Cas9 recognize and cleave DNA of incoming viruses with sequence similarity to the guide RNA (El-Mounadi et al., 2020;Garneau et al., 2010;Jinek et al., 2012;Sternberg et al., 2014). A fundamental part of the genome editing process is the identification of target genes that are linked to phenotypes of interest, such as susceptibility to abiotic stresses. ...
In this past decade, the bond between agriculture, food security, and climate change has become increasingly strong. Agriculture is recognized as one of the most endangered systems adversely affected by human activities and environmental issues. In particular, abiotic stress limits the quantity and quality of plant-based food. Heat stress, drought, and salinity impact plants at all different life stages, inducing morphological and physiological changes and provoking a reduction in their nutritional value. Accordingly, low-quality food results in a serious risk for the health of people worldwide. In this scenario, different genetic and biotechnological strategies have been investigated, including the use of New Plant Breeding Techniques (NBTs) and plant cell cultures. In this review, we describe how abiotic stresses alter the property and availability of nutritious food. In addition, we illustrate the advanced techniques that could be employed to address these issues and ameliorate the agricultural practices
... Binding of the Cas9 nuclease to the crRNA/tracrRNA complex induces a structural alteration in the protein, activating the Protospacer Adjacent Motif (PAM) recognition site. PAMs, consisting of 2 to 5 nucleotides (5'NGG3' and 5'NNGRRT3'), are essential for anchoring the Cas9 nuclease to the cleavage site [61,62]. ...
Filamentous fungi exhibit unparalleled potential as cell factories for protein production, owing to their adeptness in protein secretion and remarkable proficiency in post-translational modifications. This review delineates the role of filamentous fungi in bio-input technology across different generations and explores their capacity to generate secondary metabolites. Our investigation highlights filamentous fungi as frontrunners in the production of bioactive compounds, emphasizing the imperative nature of elucidating their metabolic repertoire. Furthermore, we delve into common strategies for genetic transformation in filamentous fungi, elucidating the underlying principles, advantages, and drawbacks of each technique. Taking a forward-looking approach, we explore the prospects of genome engineering, particularly the CRISPR-Cas9 technique, as a means to propel protein secretion in filamentous fungi. Detailed examination of the protein secretion pathways in these fungi provides insights into their industrial applications. Notably, extensive research within the scientific community has focused on Aspergillus and Trichoderma species for the industrial production of proteins and enzymes. This review also presents practical examples of genetic engineering strategies aimed at augmenting enzyme secretion in filamentous fungi for various industrial applications. These findings underscore the potential of filamentous fungi as versatile platforms for protein production and highlight avenues for future research and technological advancement in this field.
... The dCas9 and sgRNA form a DNA-recognizing complex that binds to the target DNA and sterically blocks transcription. Recognition of the target gene requires a short sequence called protospacer adjacent motif (PAM) [8][9][10] . This PAM requirement constrains targeting and affects knockdown efficiency and target flexibility. ...
Pooled knockdown libraries of essential genes are useful tools for elucidating the mechanisms of action of antibacterial compounds, a pivotal step in antibiotic discovery. However, achieving genomic coverage of antibacterial targets poses a challenge due to the uneven proliferation of knockdown mutants during pooled growth, leading to the unintended loss of important targets. To overcome this issue, we describe the construction of CIMPLE ( C RISPR i - m ediated p ooled library of e ssential genes), a rationally designed pooled knockdown library built in a model antibiotic-resistant bacteria, Burkholderia cenocepacia. By analyzing growth parameters of clonal knockdown populations of an arrayed CRISPRi library, we predicted strain depletion levels during pooled growth and adjusted mutant relative abundance, approaching genomic coverage of antibacterial targets during antibiotic exposure. We first benchmarked CIMPLE by chemical-genetic profiling of known antibacterials, then applied it to an uncharacterized bacterial growth inhibitor from a new class. CRISPRi-Seq with CIMPLE, followed by biochemical validation, revealed that the novel compound targets the peptidyl-tRNA hydrolase (Pth). Overall, CIMPLE leverages the advantages of arrayed and pooled CRISPRi libraries to uncover unexplored targets for antibiotic action.
Summary
Bacterial mutant libraries in which antibiotic targets are downregulated are useful tools to functionally characterize novel antimicrobials. These libraries are used for chemical-genetic profiling as target-compound interactions can be inferred by differential fitness of mutants during pooled growth. Mutants that are functionally related to the antimicrobial mode of action are usually depleted from the pool upon exposure to the drug. Although powerful, this method can fail when the unequal proliferation of mutant strains before exposure causes mutants to fall below the detection level in the library pool. To address this issue, we constructed an arrayed essential gene mutant library (EGML) in the antibiotic-resistant bacterium Burkholderia cenocepacia using CRISPR interference (CRISPRi) and analyzed the growth parameters of individual mutant strains. We then modelled depletion levels during pooled growth and used the model to rationally design an optimized CRISPR interference-mediated pooled library of essential genes (CIMPLE). By adjusting the initial inoculum of the knockdown mutants, we achieved coverage of the bacterial essential genome with mutant sensitization. We exposed CIMPLE to a recently discovered antimicrobial of a novel class and discovered it inhibits the peptidyl-tRNA hydrolase, an essential bacterial enzyme. In summary, we demonstrate the utility of CIMPLE and CRISPRi-Seq to uncover the mechanism of action of novel antimicrobial compounds.
Graphical abstract
... Doench J et al. [54] Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 Nature Biotechnology 11 5 Hsu P et al. [55] Development and applications of CRISPR-Cas9 for genome engineering Cell 11 6 Sternberg S et al. [56] DNA interrogation by the CRISPR RNAguided endonuclease Cas9 Nature 10 7 ...
CRISPR/Cas9 has emerged as the predominant method for genome editing due to its cost-effectiveness and broad applicability, playing a crucial role in advancing sustainable practices across various sectors. This systematic review leverages the PRISMA methodology to explore CRISPR/Cas9's impact on environmental protection, thereby supporting the Sustainable Development Goals (SDGs). Analyzing data from the Web of Science, the review found significant growth in related publications, with a 30% increase since 2014, predominantly from the US, China, Germany, and the UK. The study categorizes the scientific developments into three trends: advancements in agriculture, gene editing techniques, and biofuel production. Key discussions include the use of CRISPR/Cas9 in developing fourth-generation biofuels and environmental biosensors, as well as its applications in enhancing genetic resilience and controlling invasive species. These innovations highlight CRISPR/Cas9's potential in promoting sustainable resource management and energy generation, marking a pivotal contribution to ecological conservation and sustainability efforts.
... We first injected dCas9 gRNP into singletethered DNA curtains, and then flushed out excess protein with high salt buffer. As expected, dCas9 gRNPs preferentially bound to the target site 22 (Fig. 2a-c). In contrast, on the timescale of the experiment, dSpRY gRNPs bound non-specifically along the entire length of the DNA with no significant enrichment at the target site. ...
CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.
... The cognate PAM binding initiates DNA melting and strand switching that is facilitated by a phosphate lock loop in the PI domain. These changes introduce a kink in the TS of the target DNA to enable its base pairing with the seed region of the crRNA flanking the PAM, thereby producing a single-stranded NTS and initializing the R-loop formation (74,77,220,221). ...
... The process of Cas9 temporal binding to PAM sequences located within the target DNA is observed to initiate the melting of DNA strands upstream of the PAM region. This, in turn, occurs between 6 and 8 bases of the spacer sequence of the CRISPR RNA (crRNA) guide, leading to the formation of an R-loop, and ultimately resulting in the cleavage of the target [107,108]. Cas9 needs two small RNAs to find the target: the crRNA and the trans-activating crRNA (tracrRNA). ...
Alzheimer’s, Parkinson’s, and Huntington’s are some of the most common neurological disorders, which affect millions of people worldwide. Although there have been many treatments for these diseases, there are still no effective treatments to treat or completely stop these disorders. Perhaps the lack of proper treatment for these diseases can be related to various reasons, but the poor results related to recent clinical research also prompted doctors to look for new treatment approaches. In this regard, various researchers from all over the world have provided many new treatments, one of which is CRISPR/Cas9. Today, the CRISPR/Cas9 system is mostly used for genetic modifications in various species. In addition, by using the abilities available in the CRISPR/Cas9 system, researchers can either remove or modify DNA sequences, which in this way can establish a suitable and useful treatment method for the treatment of genetic diseases that have undergone mutations. We conducted a non-systematic review of articles and study results from various databases, including PubMed, Medline, Web of Science, and Scopus, in recent years. and have investigated new treatment methods in neurodegenerative diseases with a focus on Alzheimer’s disease. Then, in the following sections, the treatment methods were classified into three groups: anti-tau, anti-amyloid, and anti-APOE regimens. Finally, we discussed various applications of the CRISPR/Cas-9 system in Alzheimer’s disease. Today, using CRISPR/Cas-9 technology, scientists create Alzheimer’s disease models that have a more realistic phenotype and reveal the processes of pathogenesis; following the screening of defective genes, they establish treatments for this disease.
... It binds to the target DNA sequence through base pairing between the guide RNA and the complementary DNA sequence. 8 This binding triggers the activation of Cas9, leading to the generation of double-strand DNA breaks (DSBs) within the target sequence. 9 DSBs can be repaired through two primary cellular mechanisms: nonhomologous end joining (NHEJ) and homologydirected repair (HDR). ...
Introduction CRISPR–Cas9 gene editing, leveraging bacterial defense mechanisms, offers precise DNA modifications, holding promise in curing genetic diseases. This review critically assesses its potential, analyzing evidence on therapeutic applications, challenges, and future prospects. Examining diverse genetic disorders, it evaluates efficacy, safety, and limitations, emphasizing the need for a thorough understanding among medical professionals and researchers. Acknowledging its transformative impact, a systematic review is crucial for informed decision-making, responsible utilization, and guiding future research to unlock CRISPR–Cas9's full potential in realizing the cure for genetic diseases.
Methods A comprehensive literature search across PubMed, Scopus, and the Web of Science identified studies applying CRISPR–Cas9 gene editing for genetic diseases, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Inclusion criteria covered in vitro and in vivo models targeting various genetic diseases with reported outcomes on disease modification or potential cure. Quality assessment revealed a generally moderate to high risk of bias. Heterogeneity prevented quantitative meta-analysis, prompting a narrative synthesis of findings.
Discussion CRISPR–Cas9 enables precise gene editing, correcting disease-causing mutations and offering hope for previously incurable genetic conditions. Leveraging inherited epigenetic modifications, it not only fixes mutations but also restores normal gene function and controls gene expression. The transformative potential of CRISPR–Cas9 holds promise for personalized treatments, improving therapeutic outcomes, but ethical considerations and safety concerns must be rigorously addressed to ensure responsible and safe application, especially in germline editing with potential long-term implications.
... Therefore, the selection and design of active gRNA are critical for CRISPR-Cas9-mediated gene editing. However, the efficacy of guide RNA (gRNA) is impacted by various factors, encompassing gRNA structure (Abdel-Mawgoud and Stephanopoulos, 2020;Creutzburg et al., 2020;Magnusson et al., 2021;Riesenberg et al., 2022), conformational transitions Wang et al., 2019;Kim et al., 2020;Talas et al., 2021), R-loop formation (Gong et al., 2018), Cas9 and Cas12a variants Guo et al., 2018;Wang et al., 2019;Kim et al., 2020), PAM sequences (Doench et al., 2014), supercoiling (Ivanov et al., 2020), DNA covalent modification (Tao et al., 2017;Vlot et al., 2018;Liu Y et al., 2020;Dong et al., 2021), interactions at non-targeted sites (Sternberg et al., 2014;Moreb et al., 2020), target copy number (Ivanov et al., 2020), and target accessibility (Chen et al., 2016;Horlbeck et al., 2016;Yarrington et al., 2018). Despite extensive screening of large guide RNA (gRNA) libraries and the development of algorithms to predict sequence-dependent gRNA activity (Park et al., 2021;Talas et al., 2021;Xiang et al., 2021), these algorithms exhibit limitations in accurately predicting other datasets, training datasets, or variations across different species (Moreb and Lynch, 2021). ...
Corynebacterium glutamicum plays a crucial role as a significant industrial producer of metabolites. Despite the successful development of CRISPR-Cas9 and CRISPR-Cas12a-assisted genome editing technologies in C. glutamicum, their editing resolution and efficiency are hampered by the diverse on-target activities of guide RNAs (gRNAs). To address this problem, a hybrid CRISPR-Cas9-Cas12a genome editing platform (HyCas9-12aGEP) was developed in C. glutamicum in this study to co-express sgRNA (corresponding to SpCas9 guide RNA), crRNA (corresponding to FnCas12a guide RNA), or hfgRNA (formed by the fusion of sgRNA and crRNA). HyCas9-12aGEP improves the efficiency of mapping active gRNAs and outperforms both CRISPR-Cas9 and CRISPR-Cas12a in genome editing resolution and efficiency. In the experiment involving the deletion of the cg0697-0740 gene segment, an unexpected phenotype was observed, and HyCas9-12aGEP efficiently identified the responsible genotype from more than 40 genes. Here, HyCas9-12aGEP greatly improve our capability in terms of genome reprogramming in C. glutamicum.
... One potential disadvantage of off-target activity is that it could lead to genomic instability and mosaicism in animal models of disease [9]. The specificity of CRISPR/Cas9 relies on the design of single guide RNA (sgRNA) and protospacer adjacent motif (PAM) sequences [10,11], so the bioinformatic prediction of off-target sites has attracted much attention. In most primary studies, the potential off-target sites predicted by online software were analyzed by T7EN1 cleavage assays, Sanger sequencing or next-generation sequencing [2,[12][13][14]. ...
... In-vitro induction of individual PAM mutations rendered Cas9 inactive, enabling phages to bypass host defenses and infect bacteria. Cas9 probes for the target DNA, according to studies, by first confirming that the PAM is accurate and then examining the adjacent DNA for RNA complementarity (Sternberg et al., 2014). ...
... Although the exact cause of the conformational shift is unknown, Jinek and colleagues propose that it may be brought about by steric interactions or a weak connection between protein side chains and RNA bases (39). Upon activation, the Cas9 protein randomly searches for target DNA by attaching to regions that correspond to its protospacer adjacent motif (PAM) sequence (40 ...
Most medically important bacteria have genomes that contain clustered regularly
interspaced short palindromic repeats (CRISPRs) and the gene Cas that they are linked to, which operate as a protective mechanism against external invaders like plasmids and viruses. The study's objective is to find out how frequently the CRISPR/Cas system is present in Klebsiella pneumoniae clinical isolates and wild-type bacteria. Further, to detect the distribution of genes encoding for extended-spectrum β-lactamase (ESBL), and carbapenemase among CRISPR positive and negative isolates using real-time PCR. Furthermore, to determine the association between drug resistance patterns and CRISPR/Cas system.
In this cross-sectional study, the various clinical specimens were collected from
different sites of infections. A total of 176 bacterial isolates were collected (One hundred were Klebsiella pneumoniae, the other isolates were Pseudomonas species 28 isolates, E. coli 23 isolates, Acinetobacter species 13 isolates, and 12 isolates Proteus species). The study patients were admitted to the Medical City and Al Ramadi Teaching Hospitals in Iraq during the period between October 2021 and March 2022. The clinical specimens were processed
and cultured in the microbiology laboratories of these same hospitals according to standard criteria. The VITEK®2 Compact B System was used for the confirmative and final identification of Klebsiella pneumoniae isolates using VITEK®2 GN ID cards. The antimicrobial susceptibility test of the study isolates was determined via the VITEK®2 Compact B system using VITEK®2 AST-GN cards according to the manufacturer’s instructions. The antimicrobial susceptibility surveillance was expressed as minimum inhibitory concentration (MIC) values and interpreted as susceptible, intermediate, or resistant. Isolates were resistant to ampicillin (98%), followed by cefazolin (85%), ceftazidime (84%), ceftriaxone (83%), cefepime and trimethoprim-sulphamethoxazole (76%), levofloxacin (50%), cefoxitin (43%), ciprofloxacin (40%), piperacillin-tazobactam (29%), gentamicin (27%), nitrofurantoin (22%), amikacin (18%), ertapenem and imipenem (15%) in addition to tigecycline (6%). II NO45 cards (ESBL test panel) of VITEK®2 Compact B System were used to test each isolate for ESBL production. The production of ESBL was detected in 71 out of 100 study isolates (71%) of ESBL isolates. It was observed that of the total study isolates of Klebsiella pneumonia, 15 (15%) were resistant to group (ertapenem and imipenem). The other 14 (14%) were negative for the production of ESBL and carbapenemase (14/100) was sensitive to the antimicrobial agents. Further, the bacteria were classified into multidrug (77%), extensively drug-resistant (11.0%), and pandrug-resistant (4.0%).
In the molecular part of the study Cas1, Cas3, CRISPR1, CRISPR2, and CRISPR3 were
detected through amplification of their primers by conventional PCR. The prevalence of the CRISPR/Cas system was 4(26.67%) in carbapenem-resistant strains and had higher pan resistance to other antibiotics, and 21(29.58%) in the extended spectrum of β-lactamase producer isolates, while it was 8(57.14%) in drug-sensitive isolates, showing a highly significant inverse correlation between prevalence and resistance. The low frequency of the CRISPR/Cas system in drug-resistant Klebsiella pneumoniae implied that CRISPR/Cas may play a role in preventing the acquisition of drug-resistance genes. The occurrence of this system for the sensitive to antimicrobial agents isolates was 6.0 (42.86%), 1.0 (7.14%), and 1.0 (7.14%) for CRISPR1/Cas, RISPR2/Cas, and CRISPR3/Cas respectively. On the other hand, the resistance ratio for the study isolates were 6.0 (8.45%), 19.0 (26.76%), and 13.0 (18.31%) for CRISPR1/Cas, CRISPR2/Cas, and CRISPR3/Cas in ESBL isolates respectively. Also, 1.0 (6.67%), 4.0 (26.67%), and 3.0 (20.0%) for CRISPR1/Cas, CRISPR2/Cas, and CRISPR3/Cas in metallo β-lactamase producer isolates respectively were detected.
The quantitative real-time polymerase chain reaction was used to detect the carbapenemase genes (qPCR). For the detection of blaKPC, blaOXA, blaNDM, blaVIM, and blaIMP genes according to the manufacturer's instructions using (Sacace Biotechnologies S.r.l., Como, Italy) with a qPCR kit (Sacace Biotechnologies S.r.l., Como, Italy). Previous carbapenemase-producing isolates that were positive for studied genes, were used as positive controls. Among the carbapenem-resistant isolates, blaVIM and blaNDM were the primary drug resistance-associated genes and other several types of genes like blaIMP blaOXA blaKPC were also tested. Our study's findings showed that out of 15 Klebsiella pneumonia III positive isolates for metallo β-lactamase all the isolates 15 (100%) had no expression for these metallo β-lactamase genes (KPC and OXA). Also, Our study's findings showed that out
of 15 Klebsiella pneumoniae-positive isolates for metallo β-lactamase, 5 (33.33%) were confirmed as co-existence of metallo β-lactamase VIM and NDM producer isolates. In contrast, 10 (66.67%) of these isolates have not expressed these genes. The results revealed that two isolates had imipenemase encoding genes for all study isolates. Resistance to different types of drugs was higher in the absence of the CRISPR/Cas system. It is suggested that due to the low prevalence of CRISPR/Cas systems in antibiotic-resistant Klebsiella pneumoniae isolates, we conclude that these systems can offer protection against exogenous antibiotic resistance which can be acquired in the absence of CRISPR/Cas modules. This is indeed important as these systems can be developed in the future to be used as tools to fight antibiotic-resistant bacteria. The CRISPR/Cas9 system provides new
opportunities to eradicate MDR strains, as this RNA-guided DNA nuclease can specifically
cleave bacterial genes, leading to the re-sensitization of antibiotic-resistant cells.
CRISPR/Cas system works as an adaptive immune system which prevents the acquisition of resistance encoding genes like ESBLs and Metallo β-lactamase encoding genes. The CRISPR/Cas system will play an important role in the diagnosis and treatment of Klebsiella pneumoniae infections.
... The sgRNA is designed to base complementary pairing with 20 nucleotides (NTs) of the target DNA strand. The proximal seed region sequence of guide RNA (gRNA) preforms in the A-type conformation, and Cas9 protospacer adjacent motif (PAM) interaction structural domain binds to the PAM sequence and initiates local DNA strand separation (15)(16)(17). The Cas9 phosphate lock loop interacts with +1 phosphate on the DNA backbone of the adjacent PAM target strand, stabilizing the unwound target DNA strand and causing the first base to flip toward the gRNA (18). ...
... The only requirement for Cas9 binding and recognition of a locus is the recognition of a protospacer-adjacent motif (PAM) site, which varies by Cas protein being used, but is generally a 3-5bp sequence. The recognition of the PAM site allows stable R-loop formation and directs Cas9 nuclease to cut the DNA 3-5 base pairs upstream (Anders et al., 2014;Nishimasu et al., 2014;Sternberg et al., 2014;Palermo et al., 2017). While the need for an appropriate PAM site can diminish the targeting capabilities of CRISPR-Cas9, the discovery of new CRISPR-Cas proteins from different bacterial species and lab-evolved variants has greatly increased the applicability of this technology as they have different PAM recognition motifs or have virtually no necessary motif at all, allowing for the design of sgRNAs for almost any region of the genome (Friedland et al., 2015;Hu et al., 2018;Walton et al., 2020). ...
Epigenetics refers to the molecules and mechanisms that modify gene expression states without changing the nucleotide context. These modifications are what encode the cell state during differentiation or epigenetic memory in mitosis. Epigenetic modifications can alter gene expression by changing the chromatin architecture by altering the affinity for DNA to wrap around histone octamers, forming nucleosomes. The higher affinity the DNA has for the histones, the tighter it will wrap and therefore induce a heterochromatin state, silencing gene expression. Several groups have shown the ability to harness the cell’s natural epigenetic modification pathways to engineer proteins that can induce changes in epigenetics and consequently regulate gene expression. Therefore, epigenetic modification can be used to target and treat disorders through the modification of endogenous gene expression. The use of epigenetic modifications may prove an effective path towards regulating gene expression to potentially correct or cure genetic disorders.
... Cas9 cuts 3 bps upstream of the PAM sequence (5′NGG3′ in Streptococcus pyogenes) (Garneau et al. 2010) and results in a blunt end. There is an approximately 10-12 bps spacer sequence in the 3c end in the PAM-proximal region that increases specificity and is called as seed region (Sternberg et al. 2014;Jinek et al. 2012 ...
Global food security relies on agricultural crops, and their yield is affected by various phytopathogens. These phytopathogens are prevented by using chemical pesticides that poses huge threat to the environment, surrounding microflora, and health hazards. To overcome this conventional breeding techniques are used from decades to make resistant crops against various phytopathogens and have proven its potential. Regardless of these techniques, rapid generation of disease resistant plant varieties holds immense potential. The various genetic engineering tools like zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENS), and clustered regularly interspaced short palindromic repeats)/CRISPR-associated proteins (CRISPR-Cas) based systems led to the precise genetic insertions and deletions (indels) which can be deployed. CRISPR-Cas tool is the most recent and one of the precise gene editing systems which has opened up new possibilities of gene editing for crop improvement with desired traits. In this chapter, various gene editing approaches and their advancements along with the applications have been discussed.
... Our study determined that PDREs were unable to effectively turn over after substrate cleavage and such inefficient turnover has been observed with other enzymes that target DNA. The clustered regularly interspaced short palindromic repeats (CRISPR)associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target and cleave foreign DNA (Sternberg et al., 2014); SpyCas9 was determined to be a single-turnover enzyme due to its stable binding to products for many hours (Aldag et al., 2021), even after cleavage of the original DNA strands (Raper et al., 2018), which considerably impeded in vivo repair processes (Brinkman et al., 2018). Furthermore, it was shown that the complete release of cleaved products by SpyCas9 was attained only at temperatures as high as 80°C (David et al., 2022). ...
Increasing evidence suggests that DNA phosphorothioate (PT) modification serves several purposes in the bacterial host, and some restriction enzymes specifically target PT‐DNA. PT‐dependent restriction enzymes (PDREs) bind PT‐DNA through their DNA sulfur binding domain (SBD) with dissociation constants (KD) of 5 nM~1 μM. Here, we report that SprMcrA, a PDRE, failed to dissociate from PT‐DNA after cleavage due to high binding affinity, resulting in low DNA cleavage efficiency. Expression of SBDs in Escherichia coli cells with PT modification induced a drastic loss of cell viability at 25°C when both DNA strands of a PT site were bound, with one SBD on each DNA strand. However, at this temperature, SBD binding to only one PT DNA strand elicited a severe growth lag rather than lethality. This cell growth inhibition phenotype was alleviated by raising the growth temperature. An in vitro assay mimicking DNA replication and RNA transcription demonstrated that the bound SBD hindered the synthesis of new DNA and RNA when using PT‐DNA as the template. Our findings suggest that DNA modification‐targeting proteins might regulate cellular processes involved in DNA metabolism in addition to being components of restriction‐modification systems and epigenetic readers.
... The Streptococcus pyogenes Cas9 (SpCas9), the most broadly used Cas9 enzyme, has a multi-domain architecture consisting of two nuclease domains, a His-Asn-His endonuclease (called HNH) and a Holliday junction resolvase C (RuvC), a protospacer adjacent motif (PAM)-interacting (PI) domain, and a recognition lobe that mediates nucleic acid binding through three distinct subdomains, Rec1-3 ( Fig. 1A) (6,7). Upon recognition of the short PAM sequence, Cas9 locally unwinds the double-stranded DNA, and a Cas9-bound guide RNA (gRNA) forms an RNA:DNA hybrid with the target DNA strand (TS) (8). Coordinated cleavage of the TS and non-target strand then occurs via the HNH and RuvC nucleases, respectively. ...
The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.
Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes¹. We recently discovered that IS110 family elements encode a recombinase and a non-coding bridge RNA (bRNA) that confers modular specificity for target DNA and donor DNA through two programmable loops². Here we report the cryo-electron microscopy structures of the IS110 recombinase in complex with its bRNA, target DNA and donor DNA in three different stages of the recombination reaction cycle. The IS110 synaptic complex comprises two recombinase dimers, one of which houses the target-binding loop of the bRNA and binds to target DNA, whereas the other coordinates the bRNA donor-binding loop and donor DNA. We uncovered the formation of a composite RuvC–Tnp active site that spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both target and donor DNA. A comparison of the three structures revealed that (1) the top strands of target and donor DNA are cleaved at the composite active sites to form covalent 5′-phosphoserine intermediates, (2) the cleaved DNA strands are exchanged and religated to create a Holliday junction intermediate, and (3) this intermediate is subsequently resolved by cleavage of the bottom strands. Overall, this study reveals the mechanism by which a bispecific RNA confers target and donor DNA specificity to IS110 recombinases for programmable DNA recombination.
Hace 40 años comenzó el viaje de la edición genética a través de las zinc-finger nucleases (ZFN) y las nucleasas de tipo activador de la transcripción (TALENs). El camino parecía largo y lleno de obstáculos debido a los potenciales efectos secundarios y limitaciones de dichas técnicas. No obstante, en el año 2002, después de años de interrogantes en la comunidad científica con respecto a la existencia de secuencias genéticas repetidas en distintos microorganismos, se nombra a dichas secuencias como clustered regularly interspaced short palindromic repeats (CRISPR). Desde ese momento, no se ha detenido la investigación determinando sus funciones, clasificación y potenciales usos en la edición de material genético de cualquier organismo, proveyendo ventajas con respecto a las técnicas utilizadas con anterioridad. En años recientes, su aporte a la medicina ha ganado cada vez más atención, suscitando el descubrimiento de nuevas aplicaciones siempre en constante actualización. Por ello, es necesario entender qué es el sistema CRISPR-Cas desde sus bases, estando atentos a sus avances en el futuro.
Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure‐guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR‐associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo‐electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure‐guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.
Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within noncoding regions. We have adapted the CRISPR-Cas9 gene editing tool as a novel, cancer-specific killing strategy by targeting the subset of somatic mutations that create protospacer adjacent motifs (PAMs), which have evolutionally allowed bacterial cells to distinguish between self and non-self DNA for Cas9-induced double strand breaks. Whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002) showed an average of 417 somatic PAMs per tumor produced from single base substitutions. Further analyses of 591 paired T-N samples from The International Cancer Genome Consortium found medians of ∼455 somatic PAMs per tumor in pancreatic, ∼2800 in lung, and ∼3200 in esophageal cancer cohorts. Finally, we demonstrated 69–99% selective cell death of three targeted pancreatic cancer cell lines using 4–9 sgRNAs designed using the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs in either the patient's normal cells or an irrelevant cancer using WGS. This study demonstrates the potential of CRISPR-Cas9 as a novel and selective anti-cancer strategy, and supports the genetic targeting of adult cancers.
Target cancer therapy has been developed for clinical cancer treatment based on the discovery of CRISPR (clustered regularly interspaced short palindromic repeat) -Cas system. This forefront and cutting-edge scientific technique improves the cancer research into molecular level and is currently widely utilized in genetic investigation and clinical precision cancer therapy. In this review, we summarized the genetic modification by CRISPR/Cas and CRISPR screening system, discussed key components for successful CRISPR screening, including Cas enzymes, guide RNA (gRNA) libraries, target cells or organs. Furthermore, we focused on the application for CAR-T cell therapy, drug target, drug screening, or drug selection in both ex vivo and in vivo with CRISPR screening system. In addition, we elucidated the advantages and potential obstacles of CRISPR system in precision clinical medicine and described the prospects for future genetic therapy.
In summary, we provide a comprehensive and practical perspective on the development of CRISPR/Cas and CRISPR screening system for the treatment of cancer defects, aiming to further improve the precision and accuracy for clinical treatment and individualized gene therapy.
CRISPR ribonucleoproteins (RNPs) use a variable segment in their guide RNA (gRNA) called a spacer to determine the DNA sequence at which the effector protein will exhibit nuclease activity and generate target-specific genetic mutations. However, nuclease activity with different gRNAs can vary considerably, in a spacer sequence-dependent manner that can be difficult to predict. While computational tools are helpful in predicting a CRISPR effector's activity and/or potential for off-target mutagenesis with different gRNAs, individual gRNAs must still be validated in vitro prior to their use. Here, we present compartmentalized CRISPR reactions (CCR) for screening large numbers of spacer/target/off-target combinations simultaneously in vitro for both CRISPR effector activity and specificity, by confining the complete CRISPR reaction of gRNA transcription, RNP formation, and CRISPR target cleavage within individual water-in-oil microemulsions. With CCR, large numbers of the candidate gRNAs (output by computational design tools) can be immediately validated in parallel, and we show that CCR can be used to screen hundreds of thousands of extended gRNA (x-gRNAs) variants that can completely block cleavage at off-target sequences while maintaining high levels of on-target activity. We expect CCR can help to streamline the gRNA generation and validation processes for applications in biological and biomedical research.
Modern genome editing tools particularly CRISPR/Cas9 have revolutionized plant genome manipulation for engineering resilience against changing climatic conditions, disease infestation, as well as functional genomic studies. CRISPR-mediated genome editing allows for editing at a single as well as multiple locations in the genome simultaneously, making it an effective tool for polyploid species too. However, still, its applications are limited to the model crops only. Extending it to crop plants will help improve field crops against the changing climates more rapidly and precisely. Here we describe the protocol for editing the genome of a field crop Brassica juncea (mustard), an allotetraploid and important oilseed crop of the Indo-Pak Subcontinent region. This protocol is based on the Agrobacterium-mediated transformation for the delivery of CRISPR components into the plant genome using cotyledon as explants. We elaborate on steps for recovering genome-edited knockouts, for validation of the edits, as well as recovering the transgene-free edited plants through a commonly used segregating approach.
TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems.
A tool for precise, target-specific, efficient, and affordable genome editing is a dream for many researchers, from those who conduct basic research to those who use it for applied research. Since 2012, we have tool that almost fulfils such requirements; it is based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight key factors affecting its mutagenesis outcomes: (i) factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state, or Cas9 variant, and how long it remains in place after cleavage; and (ii) factors affecting the follow-up DNA repair mechanisms including mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts, and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.
CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.
Biotechnology is one of the emerging fields that can add new and better application in a wide range of sectors like health care, service sector, agriculture, and processing industry to name some. This book will provide an excellent opportunity to focus on recent developments in the frontier areas of Biotechnology and establish new collaborations in these areas. The book will highlight multidisciplinary perspectives to interested biotechnologists, microbiologists, pharmaceutical experts, bioprocess engineers, agronomists, medical professionals, sustainability researchers and academicians. This technical publication will provide a platform for potential knowledge exhibition on recent trends, theories and practices in the field of Biotechnology
Plant-parasitic nematodes (PPNs) cause immense yield loss in crop plants that ultimately affects the global crop productivity. The sedentary endoparasitic PPNs including root-knot and cyst nematodes induce feeding cells in their host plants that serves as the permanent nutrient sink for the nematodes and affect hosts’ own nutrition. During the last two decades nematode genome and transcriptome research has made considerable advancements that was translated into functional genomics studies including RNAi or host-induced gene silencing (HIGS)-based tactics deployment for obtaining nematode resistance. Nevertheless, HIGS strategy that involves targeting nematode effector genes are likely to fail because induced-silencing of effector genes (that interact with plant R genes) can result in the evolution of resistance-breaking nematode phenotypes. Recently, CRISPR-Cas9-based genome editing strategy was adopted to achieve host resistance against different phytopathogens including bacteria, fungi and virus. In those studies, researchers attempted to knock out the host susceptibility (S) genes to obtain resistance via loss of susceptibility. Since S genes are recessively inherited in the host plant, induced mutation of S genes can be durable and may confer broad-spectrum resistance. A number of S genes contributing plant susceptibility to nematodes have been characterized in different crop plants. A few of these S genes and a number of R genes were targeted for CRISPR-Cas9-based mutagenesis studies for improving nematode resistance or conferring nematode susceptibility in crop plants. However, CRISPR-Cas9-based genome editing were primarily deployed to investigate the molecular basis of plant–nematode interrelationship, rather than concentrated efforts towards targeting S genes. In this chapter, the applicability of this technique in plant–nematode infection model and its probable future outcome is being discussed in greater detail.
Eukaryotic gene expression is linked to chromatin structure and nucleosome positioning by ATP-dependent chromatin remodelers that establish and maintain nucleosome-depleted regions (NDRs) near transcription start sites. Conserved yeast RSC and ISW2 remodelers exert antagonistic effects on nucleosomes flanking NDRs, but the temporal dynamics of remodeler search, engagement, and directional nucleosome mobilization for promoter accessibility are unknown. Using optical tweezers and two-color single-particle imaging, we investigated the Brownian diffusion of RSC and ISW2 on free DNA and sparse nucleosome arrays. RSC and ISW2 rapidly scan DNA by one-dimensional hopping and sliding, respectively, with dynamic collisions between remodelers followed by recoil or apparent co-diffusion. Static nucleosomes block remodeler diffusion resulting in remodeler recoil or sequestration. Remarkably, both RSC and ISW2 use ATP hydrolysis to translocate mono-nucleosomes processively at ~30 bp/s on extended linear DNA under tension. Processivity and opposing push–pull directionalities of nucleosome translocation shown by RSC and ISW2 shape the distinctive landscape of promoter chromatin.
The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas acquired immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but it can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences, and we identify an unexpectedly versatile Cas9 protein from Neisseria meningitidis. We provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins.
Significance
Genome engineering in human pluripotent stem cells holds great promise for biomedical research and regenerative medicine, but it is very challenging. Recently, an RNA-guided nuclease system called clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) has been applied to genome engineering, greatly increasing the efficiency of genome editing. Here, using a CRISPR-Cas system identified in Neisseria meningitidis , which is distinct from the commonly used Streptococcus pyogenes system, we demonstrate efficient genome engineering in human pluripotent stem cells. Our study could have a tremendous impact in regenerative medicine.
Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.
Short guide RNAs (gRNAs) can direct catalytically inactive CRISPR-associated 9 nuclease (dCas9) to repress endogenous genes in bacteria and human cells. Here we show that single or multiple gRNAs can direct dCas9 fused to a VP64 transcriptional activation domain to increase expression of endogenous human genes. This proof-of-principle work shows that clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems can target heterologous effector domains to endogenous sites in human cells.
Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
Here, we present a simple and highly efficient method for generating and detecting mutations of any gene in Drosophila melanogaster through the use of the CRISPR/Cas9 system (clustered regularly interspaced palindromic repeats/CRISPR-associated). We show that injection of RNA into the Drosophila embryo can induce highly efficient mutagenesis of desired target genes in up to 88% of injected flies. These mutations can be transmitted through the germline to make stable lines. Our system provides at least a 10-fold improvement in efficiency over previously published reports, enabling wider application of this technique. We also describe a simple and highly sensitive method of detecting mutations in the target gene by high-resolution melt analysis and discuss how the new technology enables the study of gene function.
CRISPR-Cas systems have been used with single-guide RNAs for accurate gene disruption and conversion in multiple biological systems. Here we report the use of the endonuclease Cas9 to target genomic sequences in the C. elegans germline, utilizing single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.
The ability to artificially control transcription is essential both to the study of gene function and to the construction
of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in
the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding
activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP)
to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega
subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription
activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study
of gene networks and for the development of synthetic biology and biotechnological applications.
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.
In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator-like effector nucleases.
Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic
CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate
RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases
can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also
be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple
guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian
genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9-crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.
Ditching Invading DNA
Bacteria and archaea protect themselves from invasive foreign nucleic acids through an RNA-mediated adaptive immune system called CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated (Cas). Jinek et al. (p. 816 , published online 28 June; see the Perspective by Brouns ) found that for the type II CRISPR/Cas system, the CRISPR RNA (crRNA) as well as the trans-activating crRNA—which is known to be involved in the pre-crRNA processing—were both required to direct the Cas9 endonuclease to cleave the invading target DNA. Furthermore, engineered RNA molecules were able to program the Cas9 endonuclease to cleave specific DNA sequences to generate double-stranded DNA breaks.
Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a "seed sequence" at the 5' end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.
CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.
Specific interactions between proteins and DNA are fundamental to many biological processes. In this review, we provide a revised view of protein-DNA interactions that emphasizes the importance of the three-dimensional structures of both macromolecules. We divide protein-DNA interactions into two categories: those when the protein recognizes the unique chemical signatures of the DNA bases (base readout) and those when the protein recognizes a sequence-dependent DNA shape (shape readout). We further divide base readout into those interactions that occur in the major groove from those that occur in the minor groove. Analogously, the readout of the DNA shape is subdivided into global shape recognition (for example, when the DNA helix exhibits an overall bend) and local shape recognition (for example, when a base pair step is kinked or a region of the minor groove is narrow). Based on the >1500 structures of protein-DNA complexes now available in the Protein Data Bank, we argue that individual DNA-binding proteins combine multiple readout mechanisms to achieve DNA-binding specificity. Specificity that distinguishes between families frequently involves base readout in the major groove, whereas shape readout is often exploited for higher resolution specificity, to distinguish between members within the same DNA-binding protein family.
All immune systems must distinguish self from non-self to repel invaders without inducing autoimmunity. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci protect bacteria and archaea from invasion by phage and plasmid DNA through a genetic interference pathway. CRISPR loci are present in approximately 40% and approximately 90% of sequenced bacterial and archaeal genomes, respectively, and evolve rapidly, acquiring new spacer sequences to adapt to highly dynamic viral populations. Immunity requires a sequence match between the invasive DNA and the spacers that lie between CRISPR repeats. Each cluster is genetically linked to a subset of the cas (CRISPR-associated) genes that collectively encode >40 families of proteins involved in adaptation and interference. CRISPR loci encode small CRISPR RNAs (crRNAs) that contain a full spacer flanked by partial repeat sequences. CrRNA spacers are thought to identify targets by direct Watson-Crick pairing with invasive 'protospacer' DNA, but how they avoid targeting the spacer DNA within the encoding CRISPR locus itself is unknown. Here we have defined the mechanism of CRISPR self/non-self discrimination. In Staphylococcus epidermidis, target/crRNA mismatches at specific positions outside of the spacer sequence license foreign DNA for interference, whereas extended pairing between crRNA and CRISPR DNA repeats prevents autoimmunity. Hence, this CRISPR system uses the base-pairing potential of crRNAs not only to specify a target, but also to spare the bacterial chromosome from interference. Differential complementarity outside of the spacer sequence is a built-in feature of all CRISPR systems, indicating that this mechanism is a broadly applicable solution to the self/non-self dilemma that confronts all immune pathways.
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated CRISPR-associated sequence (CAS) proteins constitute a novel antiviral defence system that is widespread in prokaryotes. Repeats are separated by spacers, some of them homologous to sequences in mobile genetic elements. Although the whole process involved remains uncharacterized, it is known that new spacers are incorporated into CRISPR loci of the host during a phage challenge, conferring specific resistance against the virus. Moreover, it has been demonstrated that such interference is based on small RNAs carrying a spacer. These RNAs would guide the defence apparatus to foreign molecules carrying sequences that match the spacers. Despite this essential role, the spacer uptake mechanism has not been addressed. A first step forward came from the detection of motifs associated with spacer precursors (proto-spacers) of Streptococcus thermophilus, revealing a specific recognition of donor sequences in this species. Here we show that the conservation of proto-spacer adjacent motifs (PAMs) is a common theme for the most diverse CRISPR systems. The PAM sequence depends on the CRISPR-CAS variant, implying that there is a CRISPR-type-specific (motif-directed) choice of the spacers, which subsequently determines the interference target. PAMs also direct the orientation of spacers in the repeat arrays. Remarkably, observations based on such polarity argue against a recognition of the spacer precursors on transcript RNA molecules as a general rule.
Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced
short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated
(Cas) proteins from the host to mediate an antiviral response that counteracts infection. After transcription of the CRISPR,
a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing
the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable
Cascade to interfere with virus proliferation. Our results demonstrate that the formation of mature guide RNAs by the CRISPR
RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense.
In 1970 Riggs et al. (1) reported that Escherichia coli lac repressor binding to λ DNA in vitro seemed to find its target (operator) site on the DNA at a rate as much as 1000-fold faster than the upper limit estimated for a diffusion-controlled process involving macromolecules of this size. This observation startled and intrigued many physically oriented molecular biologists and biochemists and initiated a flurry of theoretical and experimental papers seeking to offer an explanation. However, scrutiny of the older literature reveals that scientists, ranging from mathematicians to biologists, had long been concerned with how systems of various sorts might transcend the rate limits set by three-dimensional diffusion control (2).
Such problems are now of interest at many different levels. The pure physical chemist feels that an understanding of such phenomena might provide new insight into what happens when molecules meet and rearrange in the course of forming and passing through the transition state complex. The enzyme mechanician hopes that the secrets of some of the astonishing increases in rates achieved in enzyme-catalyzed reactions may be revealed by a study of these rate accelerations. And the cell biologist who studies macromolecular interactions and assembly processes is intrigued by the possibility that these systems may reveal opportunities for acceleration of intracellular rates beyond the limits set by the relatively slow diffusion of macromolecules in the cytoplasm.
In this minireview we propose to touch on recent progress in all of these areas but will focus primarily on a problem that has engaged our attention over the past few years, i.e. how do protein regulators of gene expression at the transcriptional level find their regulatory DNA targets at speeds that appear to be faster than diffusion controlled?
In this paper we summarize the various factors that must be considered in establishing the operational specificity of the binding of a protein regulator of gene expression to a DNA target site. We consider informational (combinatorial) aspects of binding-site specification, actual recognition mechanisms, and the thermodynamics of target-site selection against a background of competing pseudospecific and non-(sequence)-specific DNA binding sites. The results provide insight into the design, specification, and possibly the evolution of regulatory proteins and their chromosomal binding targets, as well as into practical aspects of the design of regulatory-protein isolation schemes and physicochemical regulatory considerations in vivo.
During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 +/- 0.6 A, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force-velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site.
Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria
and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria
integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance
phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.
Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 system. Three guide RNAs (gRNAs) with a 20-22 nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer adjacent motif (PAM). The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation (insertion or deletion) by error prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome editing events, the mutation efficiency at these target sites was estimated to be 3 - 8%. In addition, off-target effect of an engineered gRNA-Cas9 was found on an imperfectly paired genomic site, but it had lower genome editing efficiency than the perfectly matched site. Further analysis suggests that mis-match position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 targeting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants.
The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.
The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.
Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.
We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.
Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.
Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.
Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer
the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus,
we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells.
We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple
gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting
~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome
engineering.
Gene expression, DNA replication and genome maintenance are all initiated by proteins that must recognize specific targets from among a vast excess of nonspecific DNA. For example, to initiate transcription, Escherichia coli RNA polymerase (RNAP) must locate promoter sequences, which compose <2% of the bacterial genome. This search problem remains one of the least understood aspects of gene expression, largely owing to the transient nature of search intermediates. Here we visualize RNAP in real time as it searches for promoters, and we develop a theoretical framework for analyzing target searches at the submicroscopic scale on the basis of single-molecule target-association rates. We demonstrate that, contrary to long-held assumptions, the promoter search is dominated by three-dimensional diffusion at both the microscopic and submicroscopic scales in vitro, which has direct implications for understanding how promoters are located within physiological settings.
The ability of proteins to locate specific targets among a vast excess of nonspecific DNA is a fundamental theme in biology. Basic principles governing these search mechanisms remain poorly understood, and no study has provided direct visualization of single proteins searching for and engaging target sites. Here we use the postreplicative mismatch repair proteins MutSα and MutLα as model systems for understanding diffusion-based target searches. Using single-molecule microscopy, we directly visualize MutSα as it searches for DNA lesions, MutLα as it searches for lesion-bound MutSα, and the MutSα/MutLα complex as it scans the flanking DNA. We also show that MutLα undergoes intersite transfer between juxtaposed DNA segments while searching for lesion-bound MutSα, but this activity is suppressed upon association with MutSα, ensuring that MutS/MutL remains associated with the damage-bearing strand while scanning the flanking DNA. Our findings highlight a hierarchy of lesion- and ATP-dependent transitions involving both MutSα and MutLα, and help establish how different modes of diffusion can be used during recognition and repair of damaged DNA.
In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary "nonself" DNA sequences for destruction, while avoiding binding to "self" sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for nonself target recognition and binding. Combining a 2.3 Å crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved three base pair motif that is required for nonself target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade.
Bacteria and archaea possess adaptive immune systems that rely on small RNAs for defense against invasive genetic elements. CRISPR (clustered regularly interspaced short palindromic repeats) genomic loci are transcribed as long precursor RNAs, which must be enzymatically cleaved to generate mature CRISPR-derived RNAs (crRNAs) that serve as guides for foreign nucleic acid targeting and degradation. This processing occurs within the repetitive sequence and is catalyzed by a dedicated Cas6 family member in many CRISPR systems. In Pseudomonas aeruginosa, crRNA biogenesis requires the endoribonuclease Csy4 (Cas6f), which binds and cleaves at the 3' side of a stable RNA stem-loop structure encoded by the CRISPR repeat. We show here that Csy4 recognizes its RNA substrate with an ~50 pM equilibrium dissociation constant, making it one of the highest-affinity protein:RNA interactions of this size reported to date. Tight binding is mediated exclusively by interactions upstream of the scissile phosphate that allow Csy4 to remain bound to its product and thereby sequester the crRNA for downstream targeting. Substrate specificity is achieved by RNA major groove contacts that are highly sensitive to helical geometry, as well as a strict preference for guanosine adjacent to the scissile phosphate in the active site. Collectively, our data highlight diverse modes of substrate recognition employed by Csy4 to enable accurate selection of CRISPR transcripts while avoiding spurious, off-target RNA binding and cleavage.
Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based bacterial immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.
Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses.
In physiological settings, nucleic-acid translocases must act on substrates occupied by other proteins, and an increasingly appreciated role of translocases is to catalyse protein displacement from RNA and DNA. However, little is known regarding the inevitable collisions that must occur, and the fate of protein obstacles and the mechanisms by which they are evicted from DNA remain unexplored. Here we sought to establish the mechanistic basis for protein displacement from DNA using RecBCD as a model system. Using nanofabricated curtains of DNA and multicolour single-molecule microscopy, we visualized collisions between a model translocase and different DNA-bound proteins in real time. We show that the DNA translocase RecBCD can disrupt core RNA polymerase, holoenzymes, stalled elongation complexes and transcribing RNA polymerases in either head-to-head or head-to-tail orientations, as well as EcoRI(E111Q), lac repressor and even nucleosomes. RecBCD did not pause during collisions and often pushed proteins thousands of base pairs before evicting them from DNA. We conclude that RecBCD overwhelms obstacles through direct transduction of chemomechanical force with no need for specific protein-protein interactions, and that proteins can be removed from DNA through active disruption mechanisms that act on a transition state intermediate as they are pushed from one nonspecific site to the next.
Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.
DNA-binding proteins survey genomes for targets using facilitated diffusion, which typically includes a one-dimensional (1D) scanning component for sampling local regions. Eukaryotic proteins must accomplish this task while navigating through chromatin. Yet it is unknown whether nucleosomes disrupt 1D scanning or eukaryotic DNA-binding factors can circumnavigate nucleosomes without falling off DNA. Here we use single-molecule microscopy in conjunction with nanofabricated curtains of DNA to show that the postreplicative mismatch repair protein complex Mlh1-Pms1 diffuses in 1D along DNA via a hopping/stepping mechanism and readily bypasses nucleosomes. This is the first experimental demonstration that a passively diffusing protein can traverse stationary obstacles. In contrast, Msh2-Msh6, a mismatch repair protein complex that slides while maintaining continuous contact with DNA, experiences a boundary upon encountering nucleosomes. These differences reveal important mechanistic constraints affecting intranuclear trafficking of DNA-binding proteins.
Single-molecule studies of biological macromolecules can benefit from new experimental platforms that facilitate experimental design and data acquisition. Here we develop new strategies to construct curtains of DNA in which the molecules are aligned with respect to one another and maintained in an extended configuration by anchoring both ends of the DNA to the surface of a microfluidic sample chamber that is otherwise coated with an inert lipid bilayer. This "double-tethered" DNA substrate configuration is established through the use of nanofabricated rack patterns comprised of two distinct functional elements: linear barriers to lipid diffusion that align DNA molecules anchored by one end to the bilayer and antibody-coated pentagons that provide immobile anchor points for the opposite ends of the DNA. These devices enable the alignment and anchoring of thousands of individual DNA molecules, which can then be visualized using total internal reflection fluorescence microscopy under conditions that do not require continuous application of buffer flow to stretch the DNA. This unique strategy offers the potential for studying protein-DNA interactions on large DNA substrates without compromising measurements through application of hydrodynamic force. We provide a proof-of-principle demonstration that double-tethered DNA curtains made with nanofabricated rack patterns can be used in a one-dimensional diffusion assay that monitors the motion of quantum dot-tagged proteins along DNA.
Here we use single-molecule imaging to determine coarse-grained intrinsic energy landscapes for nucleosome deposition on model DNA substrates. Our results reveal distributions that are correlated with recent in silico predictions, reinforcing the hypothesis that DNA contains some intrinsic positioning information. We also show that cis-regulatory sequences in human DNA coincide with peaks in the intrinsic landscape, whereas valleys correspond to nonregulatory regions, and we present evidence arguing that nucleosome deposition in vertebrates is influenced by factors that are not accounted for by current theory. Finally, we demonstrate that intrinsic landscapes of nucleosomes containing the centromere-specific variant CenH3 are correlated with patterns observed for canonical nucleosomes, arguing that CenH3 does not alter sequence preferences of centromeric nucleosomes. However, the nonhistone protein Scm3 alters the intrinsic landscape of CenH3-containing nucleosomes, enabling them to overcome the otherwise exclusionary effects of poly(dA-dT) tracts, which are enriched in centromeric DNA.
Single molecule visualization of protein-DNA complexes can reveal details of reaction mechanisms and macromolecular dynamics inaccessible to traditional biochemical assays. However, these techniques are often limited by the inherent difficulty of collecting statistically relevant information from experiments explicitly designed to look at single events. New approaches that increase throughput capacity of single molecule methods have the potential for making these techniques more readily applicable to a variety of biological questions involving different types of DNA transactions. Here we show that nanofabricated chromium barriers, which are located at strategic positions on a fused silica slide otherwise coated with a supported lipid bilayer, can be used to organize DNA molecules into molecular curtains. The DNA that makes up the curtains is visualized by total internal reflection fluorescence microscopy (TIRFM) allowing simultaneous imaging of hundreds or thousands of aligned molecules. These DNA curtains present a robust experimental platform portending massively parallel data acquisition of individual protein-DNA interactions in real time.
Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria
- G Gasiunas
- R Barrangou
- P Horvath
- V Siksnys
Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates
specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA. 2012;
109:E2579-86. [PubMed: 22949671]
RNA-programmed genome editing in human cells
- M Jinek
Jinek M, et al. RNA-programmed genome editing in human cells. Elife. 2013; 2:e00471. [PubMed:
23386978]
RNA-guided editing of bacterial genomes using CRISPR-Cas systems
- W Jiang
- D Bikard
- D Cox
- F Zhang
- L A Marraffini
Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes
using CRISPR-Cas systems. Nat Biotechnol. 2013; 31:233-239. [PubMed: 23360965]