Figure - available from: Molecular Biology Reports
This content is subject to copyright. Terms and conditions apply.
The Brief history on evolution of CRISPR/Cas system for genome editing: Discovery of CRISPR/Cas system for genome editing (1987–2012); Various approaches for eukaryotic genome editing (2013–2017); Evolution of CRISPR/Cas9 based tools (2016–2019); First clinical trial based on CRISPR/Cas9 system (2018) are represented in the roadmap
Source publication
CRISPR/Cas9 system, a bacterial adaptive immune system developed into a genome editing technology, has emerged as a powerful tool revolutionising genome engineering in all branches of biological science including agriculture, research and medicine. Rapid evolution of CRISPR/Cas9 system from the generation of double strand breaks to more advanced ap...
Citations
... 18,19 Base editors (BEs) harness the advantage of CRISPR-Cas9 by combining a Cas9-nickase targeting domain with either adenosine deaminase (ABE) or cytosine deaminase (CBE) that enables efficient, scarless single-nucleotide conversion (A>G or C>T) at the desired target site without the generation of DSBs. [20][21][22] However, a limitation of BE is their propensity to generate bystander mutations. Some bystander mutations can introduce new disease-causing variation even as the original disease-causing variant is corrected, but this can often be circumvented by careful selection of single guide RNA (sgRNA) and BE variants. ...
... Frontiers in Microbiology 04 frontiersin.org target gene at the 5′ end, followed by a repeat of crRNA and an inverted repeat of tracrRNA (Prasad et al., 2021). During gene editing, a ribonucleoprotein composed of the Cas9 protein and sgRNA recognizes the PAM and target sequence on the gene. ...
Filamentous fungi play a crucial role in environmental pollution control, protein secretion, and the production of active secondary metabolites. The evolution of gene editing technology has significantly improved the study of filamentous fungi, which in the past was laborious and time-consuming. But recently, CRISPR-Cas systems, which utilize small guide RNA (sgRNA) to mediate clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), have demonstrated considerable promise in research and application for filamentous fungi. The principle, function, and classification of CRISPR-Cas, along with its application strategies and research progress in filamentous fungi, will all be covered in the review. Additionally, we will go over general matters to take into account when editing a genome with the CRISPR-Cas system, including the creation of vectors, different transformation methodologies, multiple editing approaches, CRISPR-mediated transcriptional activation (CRISPRa) or interference (CRISPRi), base editors (BEs), and Prime editors (PEs).
... The utilization of CRISPR-Cas9 has led to an important evolution in the field of genome engineering, since it allows for accurate and effective editing of DNA sequences. (Prasad et al., 2021). This method can be utilized to disrupt, knock out (KO), or knock in genes (Nishiga et al., 2021). ...
Mesenchymal stem cells (MSCs) have garnered significant attention in biomedical studies due to their remarkable properties, such as self-renewal, differentiation into diverse cell types and immune responses. The proliferation and differentiation of MSCs are significantly influenced by the ligand-dependent transcription factor known as the aryl hydrocarbon receptor (AhR). In order to understand the roles of Ahr in adipose-derived MSCs (AD-MSCs), we disrupted the Ahr gene in this work using CRISPR/Cas9 gene editing technology. The gRNA/Cas9 dual vector and donor vector were introduced into the AD-MSC cell line (PT-5006). Green fluorescent protein (GFP) expression and puromycin resistance were used to identify the transfected cells. AhR-KO cells were cloned and confirmed by PCR and sequencing. By using RT-qPCR, the expression levels of AhR and the Ahr-related gene Cyp1B1 were investigated. The results showed that the knocked out of AhR using CRISPR/Cas9, resulting significantly decreased expression of 7.69-fold for AhR and 3.70-fold for Cyp1B1 in the cells. These cell clones and CRISPR/Cas9 vectors could be used as tools to investigate the functions of AhR in both AD-MSCs and other cell types.
... This may be related to the meroblastic cleavage of E. carinicauda zygotes in the early cleavage stage (43). Moreover, the using of Cas9 nuclease protein might also contribute to the high gene editing efficiency, since it is delivered in functional form, and it also has less toxicity compared with plasmid and mRNA (44,45). And, the use of higher concentrations of sgRNA and Cas9 nuclease protein than those used in previous studies might also be the reason for the high efficiency of gene editing (28,29,46). ...
In the culture of crustaceans, most species show sexual dimorphism. Monosex culture is an effective approach to achieve high yield and economic value, especially for decapods of high value. Previous studies have developed some sex control strategies such as manual segregation, manipulation of male androgenic gland and knockdown of the male sexual differentiation switch gene encoding insulin-like androgenic gland hormone (IAG) in decapods. However, these methods could not generate hereditable changes. Genetic manipulation to achieve sex reversal individuals is absent up to now. In the present study, the gene encoding IAG ( EcIAG ) was identified in the ridgetail white prawn Exopalaemon carinicauda . Sequence analysis showed that EcIAG encoded conserved amino acid structure like IAGs in other decapod species. CRISPR/Cas9-mediated genome editing technology was used to knock out EcIAG . Two sgRNAs targeting the second exon of EcIAG were designed and microinjected into the prawn zygotes or the embryos at the first cleavage with commercial Cas9 protein. EcIAG in three genetic males was knocked out in both chromosome sets, which successfully generated sex reversal and phenotypic female characters. The results suggest that CRISPR/Cas9-mediated genome editing technology is an effective way to develop sex manipulation technology and contribute to monosex aquaculture in crustaceans.
... CRISPR is a powerful tool that has the potential to revolutionize genomic engineering by potentially treating various diseases (1). ...
The expanding field of precision gene editing using CRISPR/Cas9 has demonstrated its potential as a transformative technology in the treatment of various diseases. However, whether this genome-editing tool could be used to modify neural circuits in the central nervous system (CNS), which are implicated in complex behavioral traits, remains uncertain. In this study, we demonstrate the feasibility of noninvasive, intranasal delivery of adeno-associated virus serotype 9 (AAV9) vectors containing CRISPR/Cas9 cargo within the CNS resulting in modification of the HTR2A receptor gene. In vitro, exposure to primary mouse cortical neurons to AAV9 vectors targeting the HT2RA gene led to a concentration-dependent decrease in spontaneous electrical activity following multielectrode array (MEA) analysis. In vivo, at 5 weeks postintranasal delivery in mice, analysis of brain samples revealed single base pair deletions and nonsense mutations, leading to an 8.46-fold reduction in mRNA expression and a corresponding 68% decrease in the 5HT-2A receptor staining. Our findings also demonstrate a significant decrease in anxiety-like behavior in treated mice. This study constitutes the first successful demonstration of a noninvasive CRISPR/Cas9 delivery platform, capable of bypassing the blood–brain barrier and enabling modulation of neuronal 5HT-2A receptor pathways. The results of this study targeting the HTR2A gene provide a foundation for the development of innovative therapeutic strategies for a broad range of neurological disorders, including anxiety, depression, attentional deficits, and cognitive dysfunction.
... Lenti-viral based approaches to complement the defective adult haemoglobin with non-sickling fetal or adult haemoglobin are currently the preferred strategies, but the long-term safety issues due to insertional mutagenesis are unknown (Pawliuk et al., 2001;Ribeil et al., 2017;Kanter et al., 2022). CRISPR/Cas based approaches are now gaining recognition as ex vivo gene editing tools owing to the efficiency and versatility (Barbarani et al., 2020;Prasad et al., 2021). ...
Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.
... CRISPR-based nucleic acid detection methods are applied in the diagnosis of COVID-19. This system is also applied in the detection of viral, fungal and bacterial pathogens, in the diagnosis of infectious diseases and various types of cancer [48]. ...
The pathogenic mechanisms of these diseases must be well understood for the treatment of neurological disorders such as Huntington's disease. Huntington's Disease (HD), a dominant and neurodegenerative disease, is characterized by the CAG re-expansion that occurs in the gene encoding the polyglutamine-expanded mutant Huntingtin (mHTT) protein. Genome editing approaches include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats/Caspase 9 (CRISPR/Cas9) systems. CRISPR/Cas9 technology allows effective gene editing in different cell types and organisms. Through these systems are created isogenic control of human origin induced pluripotent stem cells (iPSCs). In human and mouse models, HD-iPSC lines can be continuously corrected using these systems. HD-iPSCs can be corrected through the CRISPR/Cas9 system and the cut-and-paste mechanism using isogenic control iPSCs. This mechanism is a piggyBac transposon-based selection system that can effectively switch between vectors and chromosomes. In studies conducted, it has been determined that in neural cells derived from HD-iPSC, there are isogenic controls as corrected lines recovered from phenotypic abnormalities and gene expression changes. It has been determined that trinucleotide repeat disorders occurring in HD can be cured by single-guide RNA (sgRNA) and normal exogenous DNA restoration, known as the single guideline RNA specific to Cas9. The purpose of this review in addition to give general information about HD, a neurodegenerative disorder is to explained the role of CRISPR/Cas9 system with iPSCs in HD treatment.
... 26 In addition, the gene-editing tool clustered regularly interspaced short palindromic repeats (CRISPR) is boosting the development of new gene-therapy-based medicines. 27 Depending on the application, gene therapy can be used for gene augmentation (adding a gene to a cell), gene silencing (inactivation of a gene), and gene editing (revising the existing genetic code). ...
Skin injuries and chronic non-healing wounds are one of the major global burdens on the healthcare systems worldwide due to their difficult-to-treat nature, associated co-morbidities and high health care costs. Angiogenesis has a pivotal role in the wound healing process, which becomes impaired in many chronic non-healing wounds, leading to several healing disorders and complications. Therefore, induction or promotion of angiogenesis can be considered a promising approach for healing of chronic wounds. Gene therapy is one of the most promising upcoming strategies for the treatment of chronic wounds. It can be classified into three main approaches: gene augmentation, gene silencing, and gene editing. Despite the increasing number of encouraging results obtained using nucleic acids (NAs) as active pharmaceutical ingredients of gene therapy, efficient delivery of NAs to their site of action (cytoplasm or nucleus) remains a key challenge. Selection of the right therapeutic cargo and delivery methods are crucial for a favorable prognosis of the healing process. This article presents an overview of gene therapy and non-viral delivery methods for angiogenesis induction in chronic wounds.
... The target site, which is approximately 20 bp, is determined by the presence of a protospacer-adjacent motif (PAM) at the 3 -end and identified by complementary gRNA [69]. The natural diversity of PAM sequences recognized by the Cas proteins from different bacterial species considerably enhances the targeting scope of CRISPR-mediated genome editing [61,70]. ...
Polyglutamine (polyQ) diseases, including Huntington’s disease, are a group of late-onset progressive neurological disorders caused by CAG repeat expansions. Although recently, many studies have investigated the pathological features and development of polyQ diseases, many questions remain unanswered. The advancement of new gene-editing technologies, especially the CRISPR-Cas9 technique, has undeniable value for the generation of relevant polyQ models, which substantially support the research process. Here, we review how these tools have been used to correct disease-causing mutations or create isogenic cell lines with different numbers of CAG repeats. We characterize various cellular models such as HEK 293 cells, patient-derived fibroblasts, human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs) and animal models generated with the use of genome-editing technology.
The discovery of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas technology has transformed the science of genome editing, providing exemplary accuracy and efficiency in changing DNA sequences. It has generated a resurgence in genetic engineering that offers benefits in various sectors, including basic scientific research and translational medicine. By allowing us to alter, discover, and identify certain gene sequences, these genome editing techniques have expanded the possibilities for scientific investigation. The CRISPR–Cas9 system has gained significant acceptance throughout the research community in a very short period due to its extraordinary simplicity, resilience, and versatility. Its uses span various activities, including the construction of transgenic animals and plants, the clarification of disease processes, the discovery of disease targets, the generation of model cell lines, and the control of gene expression. This technology also emphasizes how crucial it is to monitor its development closely. Starting with its invention and emphasizing significant developments in the realm of drug discovery, this review article seeks to offer a thorough overview of the present status of CRISPR–Cas technology in a tailored style, future trends, their opportunities, challenges, and ethical issues that could arise from the continuous advancement and use of this technology.
