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TAL effector RVD specificities and efficiencies
... TALENs predicted using TALEN targeter web-tool were screened at multiple levels. Total predicted TALENs were first screened to follow Streubel"s guidelines of TALE RVD specificities and efficiencies (Streubel et al., 2012). Further, TALENs were screened for their target having more than 40% GC content. ...
... Of total 333 TALEN pairs designed, 153 pairs of TALENs following Streubel"s guidelines were retained for further analyses whereas remaining 180 pairs were excluded from the study. This screening was done to achieve high specificity and efficiency of TALENs (Streubel et al., 2012). ...
... Filtration of TALENs on the basis of Streubel et al. (2012) guidelines selects/screens highly active TALENs containing properly spaced strong RVDs. Further, these guidelines filter TALENs to have strong RVDs: HD against C nucleotide and NH against G nucleotide. ...
Bacterial blight disease in rice is caused by Xanthomonas oryzae pv. oryzae (XOO) through binding of type III secretion system effectors to susceptibility genes in host. PthXo1 effectors secreted by XOO binds to promoter region of sugar transporter gene Os8N3 thereby activating dominant allele of Xa13 susceptibility gene in rice. Introgression of recessive xa13 allele by molecular breeding programs have successfully imparted resistance against PthXo1 effector mediated disease in few rice cultivars. However, molecular breeding mediated introgression of disease resistance in hundreds of susceptible cultivars is a daunting task. Recent advancements in the field of genome editing technology by use of engineered nucleases ZFN, TALEN and CRISPR-Cas9 system allow fast and précised modifications in the genome. Therefore, the present study focus on designing of Xa13 locus specific TALENs for introducing bacterial blight resistance in Indica rice through TALEN mediated genome editing. Total three hundred thirty-three TALENs were designed against promoter region of Xa13. Out of these, One-hundred thirty-nine pairs of TALENs that follow Streubel's guidelines and having targets with >40% GC content were retained. Further, Screening of these selected TALENs on basis of distance of their putative cleavage site from PthXo1 effectors binding site in rice genome resulted into eleven TALEN pairs. These eleven TALEN pairs were further screened for their number of putative targets in host genome and on the TAL score. Finally only three pairs possess a unique target site and score below a cutoff of four. Best scoring TALEN pair was selected for designing of TALEN coding gene sequences codon-optimized for high level expression in rice. These TALEN coding genes can be used for introducing deletions in Xa13 promoter and impart resistance against bacterial blight disease in rice.
... The promoter-binding specificity of TALEs is determined by repeat units. Each repeat unit has near-perfect 33 to 35 amino acid repeats, with two variable amino acids at position 12 and 13 (termed the 'RVD', for repeat-variable 2 of 9 diresidue) [12,13]. TALE-encoding genes (Tal genes) are presumed to be highly dynamic and varied in RVDs and have greatly contributed to the production of new toxicity and the escape of plant immunity by Xoo [14,15]. ...
... The promoter-binding specificity of TALEs is determined by repeat units. Each repeat unit has near-perfect 33 to 35 amino acid repeats, with two variable amino acids at position 12 and 13 (termed the 'RVD', for repeat-variable diresidue) [12,13]. TALE-encoding genes (Tal genes) are presumed to be highly dynamic and varied in RVDs and have greatly contributed to the production of new toxicity and the escape of plant immunity by Xoo [14,15]. ...
Xanthomonas oryzae pv. oryzae (Xoo) is a causative agent of rice bacterial blight (BB). In 2020–2022, BB re-emerged, and there was a break out in the Yangtze River area, China. The pandemic Xoo strain, LA20, was isolated and identified from cultivar Quanyou1606 and demonstrated to be the Chinese R9 Xoo strain, which is able to override the widely adopted xa5-, Xa7- and xa13-mediated resistance in rice varieties in Yangtze River. Here, we report the complete genome of LA20 by PacBio and Illumina sequencing. The assembled genome consists of one circular chromosome of 4,960,087 bp, sharing 99.65% sequence identity with the traditional representative strain, YC11 (R5), in the Yangtze River. Comparative genome analysis of LA20 and YC11 revealed the obvious variability in Tal genes (the uppermost virulence determinants) in numbers and sequences. Particularly, six Tal genes were only found in LA20, but not in YC11, among which Tal1b (pthXo1)/Tal4 (pthXo6), along with the lost one, pthXo3 (avrXa7), might be the major factors for LA20 to overcome xa5-, Xa7- and xa13-mediated resistance, thus, leading to the resurgence of BB. This complete genome of the new pandemic Xoo strain will provide novel insights into pathogen evolution, the traits of pathogenicity on genomic level and the epidemic disease status in China.
... However, although each RVD typically favors one particular base, there is some promiscuity in binding the DNA. This means that usually more than one base can be recognized by a given RVD (Streubel et al., 2012;Guilinger et al., 2014;Juillerat et al., 2014). To induce a DNA double strand break (DSB), a TALEN pair is designed such that either DBD targets an opposing DNA strand of the target site in a tail-to-tail configuration, separated by a spacer of 10-25 bps (Mussolino et al., 2011;Christian et al., 2012). ...
... While CAST-Seq based detection of chromosomal rearrangements is agnostic to the designer nuclease platform, the nomination of the off-target sites is not. The annotation of TALEN off-target sites is particularly challenging because TALE RVDs are-despite showing a strong preference for a particular base-rather promiscuous in their DNA binding behavior (Miller et al., 2011;Streubel et al., 2012;Guilinger et al., 2014;Juillerat et al., 2014). In order to improve the reliability of TALEN off-target site nomination, we took advantage of the fact that the CAST-Seq read coverage at a given chromosomal region is a strong indicator for the presence of an off-target site. ...
Transcription activator-like effector nucleases (TALENs) are programmable nucleases that have entered the clinical stage. Each subunit of the dimer consists of a DNA-binding domain composed of an array of TALE repeats fused to the catalytically active portion of the FokI endonuclease. Upon DNA-binding of both TALEN arms in close proximity, the FokI domains dimerize and induce a staggered-end DNA double strand break. In this present study, we describe the implementation and validation of TALEN-specific CAST-Seq (T-CAST), a pipeline based on CAST-Seq that identifies TALEN-mediated off-target effects, nominates off-target sites with high fidelity, and predicts the TALEN pairing conformation leading to off-target cleavage. We validated T-CAST by assessing off-target effects of two promiscuous TALENs designed to target the CCR5 and TRAC loci. Expression of these TALENs caused high levels of translocations between the target sites and various off-target sites in primary T cells. Introduction of amino acid substitutions to the FokI domains, which render TALENs obligate-heterodimeric (OH-TALEN), mitigated the aforementioned off-target effects without loss of on-target activity. Our findings highlight the significance of T-CAST to assess off-target effects of TALEN designer nucleases and to evaluate mitigation strategies, and advocate the use of obligate-heterodimeric TALEN scaffolds for therapeutic genome editing.
... Then the parameters were adjusted to hide redundant TALENs in output. Other than these, guidelines by Streubel et al. (2012) were applied in the analysis. From the output, several TALEN targets having highest percentage of 'HD or NH' RVDs in the respective TALENs and having at least one unique restriction site at the spacer region were selected for further analyses. ...
... Then for each gene, several TALEN targets with higher percentage of HD or NH in their RVDs and having at least one unique restriction site at the spacer region were selected. TALENs having high percentage of HD or NH were selected because the binding specificity and the efficiency are higher when the percentage of HD or NH is high (Streubel et al., 2012). The selection of target sites with unique restriction sites is beneficial in experimental identification of TALEN activity (Doyle et al., 2012). ...
The human pathogens, Epstein Barr virus, human papilloma virus, herpes simplex virus-2, hepatitis B virus and Leishmania species can cause persistent infections which cannot be cured with currently available treatments. The modern gene editing techniques, Transcription Activator Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeat / CRISPR Associated protein 9 (CRISPR/Cas9) are potential candidates for the treatment of them. In this study, target sites for TALEN and CRISPR/Cas9 were identified in silico on selected essential and indispensable genes of the above pathogens targeting the ceasing of the essential functions and hence to cure the infection. The gene sequences of the pathogens were obtained from public databases and conserved sequences were identified. Then potential TALEN target sites were identified and for some selected targets, the off-target effects on the genomes of human, mouse, same pathogen and other organisms were tested and the putative functions of the mutated proteins were predicted. TALEN targets without having potential off-target effect and not leading to mutated proteins with undesirable functions were selected for each gene. The potential CRISPR/Cas9 targets without off-target effect on human and murine genomes were identified and other off-target effects were evaluated. Results showed that potential TALEN and/or CRISPR/Cas9 targets with higher binding specificity and efficiency were available for the selected genes. It can be concluded that the selected targets can potentially be used to produce respective proteins and in vitro and in vivo applications are potentially possible.
... repeats performing an α helix-random coil-α helix structure are nearly identical, except the polymorphic 12th and 13th residues, which are known as the repeat variable di-residue (RVD) and specifically specify a single binding site nucleotide through direct interactions [31][32][33][34]. The specificity and affinity for each RVD to a nucleotide were systemically studied [35,36]. Theoretically, merely four kinds of repeat, each with RVDs recognizing G, A, T, and C, respectively, are necessary to construct a DNA-binding domain specific to any given sequence. ...
... The first residue in each RVD (the 12th of the repeat) orients away from DNA to interact with the backbone of the eighth residue of the repeat to stabilize the interhelical loop and allow the second residue of the RVD to project into the major groove of the DNA and make sequence-specific contact with a single nucleotide of the sense strand [42]. The most common RVDs are HD, NG, NN, and NI for C, T, G > A, and A, respectively, in which NN can be replaced by NH for NH is more specific to G but has less affinity [34][35][36]43]. Online tools for custom TALEs and TALENs, such as TALE-NT 2.0, designation and modular assembly methods relying on Golden Gate cloning have been developed, enabling researchers to make constructs in a few days [44][45][46][47][48]. ...
Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H840A) and RuvC nuclease activity null mutant (D10A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D10A), Cas9(H840A), and Cas9(D10A, H840A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.
... HD and NG are associated with cytosine (C) and thymine (T) respectively. These associations are strong and exclusive [45]. ...
... Therefore, it is recommended to use RVD NH which binds with G with medium affinity. It is also worth noting that the binding affinity of TALE is influenced by the methylation status of the target DNA sequence [45]. A typical TALEN system usually consists of 18 repeats of 34 amino acids. ...
Background
Genome of an organism has always fascinated life scientists. With the discovery of restriction endonucleases, scientists were able to make targeted manipulations (knockouts) in any gene sequence of any organism, by the technique popularly known as genome engineering. Though there is a range of genome editing tools, but this era of genome editing is dominated by the CRISPR/Cas9 tool due to its ease of design and handling. But, when it comes to clinical applications, CRISPR is not usually preferred. In this review, we will elaborate on the structural and functional role of designer nucleases with emphasis on TALENs and CRISPR/Cas9 genome editing system. We will also present the unique features of TALENs and limitations of CRISPRs which makes TALENs a better genome editing tool than CRISPRs.
Main body
Genome editing is a robust technology used to make target specific DNA modifications in the genome of any organism. With the discovery of robust programmable endonucleases-based designer gene manipulating tools such as meganucleases (MN), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein (CRISPR/Cas9), the research in this field has experienced a tremendous acceleration giving rise to a modern era of genome editing with better precision and specificity. Though, CRISPR-Cas9 platform has successfully gained more attention in the scientific world, TALENs and ZFNs are unique in their own ways. Apart from high-specificity, TALENs are proven to target the mitochondrial DNA (mito-TALEN), where gRNA of CRISPR is difficult to import. This review talks about genome editing goals fulfilled by TALENs and drawbacks of CRISPRs.
Conclusions
This review provides significant insights into the pros and cons of the two most popular genome editing tools TALENs and CRISPRs. This mini review suggests that, TALENs provides novel opportunities in the field of therapeutics being highly specific and sensitive toward DNA modifications. In this article, we will briefly explore the special features of TALENs that makes this tool indispensable in the field of synthetic biology. This mini review provides great perspective in providing true guidance to the researchers working in the field of trait improvement via genome editing.
... A directed evolution approach yielded variants of the TALE N-terminal domain that recognize all bases, thus simplifying the positioning of TALEs and TALEN [33]. Repeat specificities can be chosen freely to match any desired target sequence, however, it is recommended to include at least 2 or 3 Cs or Gs in a TALE or TALEN target site and incorporate the RVDs HD and NN, respectively [34]. Both are considered "strong RVDs" and a TALE without any such strong RVDs can be highly inefficient [34]. ...
... Repeat specificities can be chosen freely to match any desired target sequence, however, it is recommended to include at least 2 or 3 Cs or Gs in a TALE or TALEN target site and incorporate the RVDs HD and NN, respectively [34]. Both are considered "strong RVDs" and a TALE without any such strong RVDs can be highly inefficient [34]. Since the numbers of repeats in a TALEN can be adjusted relatively freely, it is possible to place a TALEN pair to cut precisely at a desired nucleotide in a target DNA sequence. ...
TALEN were the first easy-to-use genome editing technology and sparked the genome editing revolution. Their application in multiple species brought targeted mutagenesis to the attention of scientists worldwide. Key breakthrough successes of genome editing have since been achieved using TALEN, among these, the first commercialization of an edited crop and the first human cured from cancer. TALEN have since been largely replaced by the CRISPR technologies which are somewhat easier to build, much easier to multiplex, and have spawned multiple derived techniques. Nevertheless, the flexible and precise positioning of TALEN is unmatched, and thus they have continued to evolve to new functionalities. Here, we assemble essential facts, design guidelines as well as important past and exciting novel developments.
... ZFNs employ DNA binding domain containing several zinc -finger fused with Fok1 (restriction enzyme) (Li et al. 2011) with each finger it targets 3-4 bases ( Fig. 2b) (Gupta and Musunuru 2014;Khalil 2020). Transcription activatorlike effector nuclease (TALEN), derived protein in plant pathogenic bacteria (Sun and Zhao 2013), with a novel DNA binding domain, which is structured as TALE repeats of 10 to 30 base each comprises of 33-35 amino acids in a tandem array which has variable region of two amino acid residues called repeat variable di-residue (RVD) to recognize and bind to specific sites ( Fig. 2c) (Moscou and Bogdanove 2009;Boch et al. 2009;Bogdanove and Voytas 2011;Streubel et al. 2012;Cong et al. 2013). Over ZFNs which recognize three nucleotides in one zinc finger domain, whereas TALENs has more specificity in recognizing one amino acid by each domain (Khan 2019). ...
Changes in climatic conditions increase the frequency of severity caused by abiotic stress. Understanding the physiological responses to abiotic stress is crucial for developing action plans to increase stress tolerance in plants, whether through classical breeding, genetic engineering, or other innovative approaches. Gene editing in plants is a quickly advancing field that involves the targeted modification of plant genomes to achieve specific traits or characteristics. One of the plants’ most extensively used gene-editing technologies is Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR-Cas9). CRISPR-Cas9 allows making precise changes to the DNA of plants by introducing targeted mutations. Efforts to address these challenges involve the development of stress-tolerant plant varieties through breeding, genetic engineering, and gene editing. These approaches aim to increase the ability of plants to withstand and recover from abiotic stress, ultimately improving crop resilience, quality, and yield in challenging environments. Additionally, sustainable agricultural practices and precision farming techniques can be employed to optimize resource use and mitigate the impact of abiotic stresses on crop production.
... Common susceptibility genes which are targets for TALEs include sugar transporters or transcription factors encoding genes (Boch et al. 2014;XU et al. 2017). The specificity of TALEs is determined by the sequence of repeat-variable diresidues (RVDs; Streubel et al. 2012). Accordingly, plants have evolved certain R-genes, called the executor genes (E-genes), which trap these effectors by mimicking the promoters of susceptibility genes. ...
Plant’s survival is under constant threat due to attacks by several pests and pathogens. Being sessile, plants cannot evade such attacks but only protect themselves against these invaders. The defence can be either constitutive through pre-formed physical and chemical barriers or inducible through the expression of genes related to plant defense. The latter is called the secondary line of defense, which can further occur in layers. The first layer, Pathogen Triggered Immunity (PTI), is triggered by the recognition of certain pathogen associated molecular patterns by the pattern recognition receptors on the host cell membrane. It usually entails physiological changes, production of phytoalexins and reactive oxygen species, and manifestation of hypersensitive response. Pathogens have developed strategies to overcome PTI, and they directly secrete effectors into host cells, concurrent to which plants have evolved Effector Triggered Immunity (ETI), which is the second layer of defense and involves the expression of resistance genes (R-genes). The first ever R-gene was cloned from Zea mays following where several others were cloned from model and crop plants which have revealed the occurrence of several conserved domains in the resistance proteins facilitating identification of resistance gene analogues (RGAs) using various tools and techniques. Further, the mechanisms by which products of R-genes confer resistance have been divided into nine types, broadly, through perception of pathogen effectors or due to loss-of-susceptibility. The chapter discusses the structure of R-proteins and how these conserved domains can be used for genome wide identification of RGAs. We also discuss, with examples from different plant-pathogen systems, the nine mechanisms of disease resistance in detail.
... However, it is advisable to integrate a minimum of 2 or 3 Cs or Gs in a TALE or TALEN target site and to include the RVDs HD and NN, respectively. Both of these are classified as "strong recognition variable domains" (RVDs), and a TALE lacking either of these strong RVDs may exhibit significant inefficiency [65]. The flexibility in adjusting the number of repetitions inside a TALEN allows for the exact placement of a TALEN pair to cleave a specific nucleotide within a target DNA sequence. ...
Precision genome editing is a rapidly evolving field in gene therapy, allowing for the precise modification of genetic material. The CRISPR and Cas systems, particularly the CRISPR-- Cas9 system, have revolutionized genetic research and therapeutic development by enabling precise changes like single-nucleotide substitutions, insertions, and deletions. This technology has the potential to correct disease-causing mutations at their source, allowing for the treatment of various genetic diseases. Programmable nucleases like CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs) can be used to restore normal gene function, paving the way for novel therapeutic interventions. However, challenges, such as off-target effects, unintended modifications, and ethical concerns surrounding germline editing, require careful consideration and mitigation strategies. Researchers are exploring innovative solutions, such as enhanced nucleases, refined delivery methods, and improved bioinformatics tools for predicting and minimizing off-target effects. The prospects of precision genome editing in gene therapy are promising, with continued research and innovation expected to refine existing techniques and uncover new therapeutic applications.
... Специфичность повтора может быть выбрана свободно, чтобы соответствовать любой желаемой целевой последовательности, однако рекомендуется включать по крайней мере 2 или 3 C или G в целевой сайт TALE или TALEN и включать RVD HD и NN соответственно. Оба считаются сильными RVD, и TALE без таких сильных RVD может быть крайне неэффективным [14]. Поскольку количество повторов в TALEN можно регулировать относительно свободно, можно использовать пару TALEN для точного разрезания нужного нуклеотида в целевой последовательности ДНК. ...
In this paper, we propose a new approach to the in-silico prediction of any possible DNA binding sites for the user-defined artificial TALENs. This approach based on the exponential moving average model and developed as an online service TANDIS. The direct validation of our prediction model based on the direct matching with the known results of the certain in-vitro experiments, while for the verification of its accuracy we use comparative analysis against other similar popular services like TALE-NT and TALENoffer. So thus, we have found out that the exponential moving average model brings very good results comparable with those of the Markov chain model used in TALENoffer, but TANDIS can do it much more easily because its model is much simpler. The TALE-NT prediction is even faster than ours for it has an utmost simple position-independent scoring system and drastically simplified filtering rules for the case of paired TALEs, which makes however, on the other hand, the results of such TALE-NT 's prediction much less competitive. Besides being the compromise between accuracy and efficiency, the exponential moving average model has only five parameters, so in future, it could be easily used for more intense prediction, and probably later, it can be used to cast some light on our understanding of real physical principles of the attractive interaction between a certain TALE and a random DNA site.
... Each repeat of the binding domain interacts with the DNA at the 12 and 13 amino acid residues called the repeat variable di-residues (RVDs). To arrange artificial TALE arrays, the commonly used RVDs are NI for adenine, NN or HN for guanine or adenine, HD for cytosine, and NG for thymine (Boch et al. 2009;Moscou and Bogdanove 2009;Streubel et al. 2012;Cong et al. 2013). The TALE repeat identifies only a single base pair, unlike the ZINC fingers, which recognize triplets with no site overlaps from adjoining domains (Mak et al. 2012). ...
... The commonly used TALEN system bears two TALEN units and a typical TALEN unit comprises of DNA binding TALE repeat domains (arrays of highly conserved 30-33 amino acid repeats) with flanking N and C terminal domains and one FokI nuclease catalytic domain. Individual TALE repeat can bind to a single specific DNA sequence (A/G/T/C) but the specificity is determined by two hyper variable amino acid residues present on position 12 and 13 (Table 1; Streubel et al., 2012). These two variable positions are known as Repeat Variable Diresidue (RVD). ...
... The common RVDs mainly include HD, NG, NI, and NN, which recognize cytosine (C), thymine (T), adenine (A), and adenine/guanine (A/G), respectively. Subsequently, by biochemical assays on the artificial TALE array consisting of naturally absent RVDs, the decoding of TALE was fully resolved, including all 400 (20×20) possible RVDs, which provided specific RVDs for each base, with recognition of G by NH [52,53]. ...
In recent decades, gene-editing technologies, typically based on deoxyribonucleases to specifically modify genomic sequences, have dramatically remodeled various aspects of life sciences, including fundamental research, breeding, and medical therapeutics. So far, four types of endonucleases have been adopted and optimized as gene-editing tools: meganuclease, ZFN, TALEN, and Cas nuclease from the CRISPR-Cas system. Each tool comes with its own advantages and limitations. Over the last ten years, RNA-guided Cas nucleases have been extensively investigated and successfully implemented in almost all mammalian cells due to their remarkable editing efficacy, high specificity, and flexibility in targeting the specific locus. Diverse Cas nuclease, together with meganuclease, ZFN, and TALEN, represent the key strategies for nuclease-based gene editing. However, systematic introductions and comparisons among four types of nucleases are not yet available. Here, we overview the capabilities of four types of nucleases along the development history of gene editing and describe the molecular mechanisms of substrate recognition and cleavage. Particularly, we summarize the promising CRISPR-Cas systems as well as modified tools applied for gene editing in the eukaryotic genome. Moreover, how the re-modulated nucleases and other nucleases, either naturally occurring or AI-designed, might manipulate DNA sequences is discussed and proposed.
... In the DNA binding domain consisting of 34 amino acids, there is a variable region of 2 amino acid residues called repeat variable di-residues (RVD), which can confer specificity to one of any four nucleotide bps 14,59 . Among the most common RVDs in TAL effectors are HD, NG and NI, which specify the nucleotides C, T and A respectively; these RVDs are only infrequently associated with other bases 6 . ...
Human beings today have been able to pursue goals with non-random changes in the genome of living organisms. This is the process of genetic engineering or genome editing, which involves replacing, inserting, or deleting an arbitrary genomic sequence using an artificial restriction enzyme capable of cleaving a specific part of the genome. The ability to manipulate and modify genes and functional studies as well as awareness of the molecular basis of diseases and the development of new and targeted therapies with these techniques have been created. To date, different techniques have now been developed with the ability to edit targeted genomics including Zinc-finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALEN) and Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated protein 9 (Cas9) (CRISPR/Cas9). The fusing of a zinc-finger DNA-binding domain to a DNA-cleavage domain produces artificial restriction enzymes known as Zinc-finger nucleases (ZFNs). Zinc finger domains can be designed to target a specific DNA sequence which causes ZFNs to target unique sequences in complex genomes. TALENs are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain to cut DNA strands at specific locations. CRISPR/Cas9 is a bacterial immune system against viruses in which the Cas9 nucleus connects with a single-strand guide to a complementary target sequence and makes changes. In this study, we will review the evolution and various aspects of each one over time.
... L'étude de Boch et al., en 2009 a d'ailleurs permis de déchiffrer le code des domaines TALE.Ainsi, les di-résidus variables les plus retrouvés dans les TALE sont les résidus NI (Asn-Ile), HD (His-Asp), NK (Asn-Lys)/NH (Asn-His) et NG (Asn-Gly) qui vont reconnaitre respectivement les nucléotides A, C, G et T(Boch et al., 2009) (Figure 18.B). Le di-résidu NN (Asn-Asn) présente la particularité de reconnaitre deux nucléotides, à savoir A et G, même s'il reconnaitra 50 préférentiellement le nucléotide A. Bien que présentant une spécificité pour le nucléotide G, les di-résidus NK et NH sont en général moins utilisés que les di-résidus NN qui ont une plus forte affinité pour le nucléotide G. Ces résidus peuvent malgré tout être utilisés pour avoir une plus forte spécificité(Boch et al., 2009 ;Streubel et al., 2012) (Figure 18.B et C). ...
Les DIPG (Diffuse Intrinsic Pontine Glioma) sont des tumeurs cérébrales pédiatriques au pronostic des plus sombres, et ce notamment du fait de la résistance des cellules aux différents traitements de chimio et de radiothérapie. Une des caractéristiques majeures des cellules de DIPG est qu’elles sont quasi systématiquement porteuses d’une mutation mono-allélique de l’histone H3 au niveau de sa lysine 27, et ce majoritairement sur le variant d’histone H3.3. Cette mutation, H3.3K27M, inhibe la triméthylation en K27 de l’histone H3 (H3K27me3) par un effet dominant négatif, ayant pour conséquence une réorganisation de la chromatine et ainsi une modification profonde de l’expression des gènes. Actuellement, et bien que les mutations H3K27M soient décrites comme étant un élément « driver » dans la genèse des DIPG, leur implication dans la résistance aux traitements n’a toutefois pas été pleinement établie. Afin de décrypter finement les implications de la mutation H3.3K27M, l’établissement de modèles cellulaires complémentaires d’induction mais aussi de réversion de la mutation apparaissait nécessaire.Dans ce contexte, j’ai, par transfection plasmidique, induit la mutation H3.3K27M dans trois lignées cellulaires de gliome pédiatrique sustentoriel initialement non mutées. De façon complémentaire, j’ai créé un modèle de réversion de la mutation dans des cellules de DIPG mutées H3.3K27M par la mise en place d’une stratégie utilisant le système CRISPR/Cas9, générant une cassure double brin au niveau du site de la mutation, combinée à une approche de « gene trapping » visant à restaurer la forme sauvage du gène H3F3A. Ces deux stratégies d’induction et de réversion de la mutation nous ont permis de constituer un ensemble de modèles cellulaires disponible avec et sans la mutation H3.3K27M. Fort de ceux-ci, j’ai pu entreprendre d’évaluer le rôle exact de la mutation H3.3K27M sur la résistance aux traitements de chimio et radiothérapie, la croissance cellulaire ou encore les propriétés clonogéniques.Concernant le modèle d’induction, les effets épigénétiques liés à la mutation étaient confirmés au sein des trois lignées cellulaires établies. La présence de la mutation avait alors un effet sur la croissance cellulaire dans deux des trois lignées cellulaires, et ce concomitamment avec un pouvoir clonogénique accru par l’introduction de la mutation. Ces mêmes lignées cellulaires présentaient une résistance supérieure à la radiothérapie et un screening de chimiothérapies permettait de mettre en évidence plusieurs composés pour lesquels la mutation H3.3K27M conférait une résistance. D’un point de vue plus global, il semblerait que la mutation confère un caractère agressif et résistant principalement dans un contexte de gliome de bas grade. En parallèle, j’ai pu valider la création d’un modèle de réversion de la mutation dans une lignée cellulaire de DIPG originellement mutée. Dans celui-ci, les cellules ne diffèrent que par l’absence de la mutation et présentent un retour de la marque H3K27me3. Une évaluation préliminaire des effets de la réversion de la mutation tendait à confirmer ceux obtenus avec le modèle d’induction.Le décryptage des mécanismes sous-jacents aux effets biologiques observés, nous permettra d’évaluer et de comprendre pleinement le rôle de la mutation H3.3K27M dans l’agressivité et la résistance aux traitements des DIPG et d’identifier de possibles nouvelles stratégies de traitement des gliomes malins pédiatriques du tronc cérébral.
... Another type of nucleases, TALENs, with DNA binding domains, was also employed to engineer genomic changes (Boch and Bonas 2010). Thirty-four tandem repeats are typically present in the DNA binding domain along with repeat-variable di-residue (RVD) comprised of two amino acids at positions 12 and 13, providing the TALENs with the ability to identify the intended target DNA sequence (Cong et al., 2012;Streubel et al., 2012). Like ZFNs, TALENs also introduce DSBs in the intended genomic DNA sequences, completely disrupting the gene and (or) introducing mutations. ...
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system was initially discovered as an underlying mechanism for conferring adaptive immunity to bacteria and archaea against viruses. Over the past decade, this has been repurposed as a genome-editing tool. Numerous gene editing-based crop improvement technologies involving CRISPR/Cas platforms individually or in combination with next-generation sequencing methods have been developed that have revolutionized plant genome-editing methodologies. Initially, CRISPR/Cas nucleases replaced the earlier used sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), to address the problem of associated off-targets. The adaptation of this platform led to the development of concepts such as epigenome editing, base editing, and prime editing. Epigenome editing employed epi-effectors to manipulate chromatin structure, while base editing uses base editors to engineer precise changes for trait improvement. Newer technologies such as prime editing have now been developed as a “search-and-replace” tool to engineer all possible single-base changes. Owing to the availability of these, the field of genome editing has evolved rapidly to develop crop plants with improved traits. In this review, we present the evolution of the CRISPR/Cas system into new-age methods of genome engineering across various plant species and the impact they have had on tweaking plant genomes and associated outcomes on crop improvement initiatives.
... 69 For constructing synthetic TALE arrays, the most often employed RVDs are NI for adenine, HD for cytosine, NG for thymine, and NN or HN for guanine or adenine. 70,71 TALEs are commonly built to recognize 12-to 20-bps of DNA, with more bases resulting in greater genome-editing specificity. 72 TALE DNAbinding domains may be built in a variety of ways, the easiest being Golden Gate assembly. ...
Diabetes is a metabolic disease characterized by chronic hyperglycemia. Polygenic diabetes, which encompasses type-1 and type-2 diabetes, is the most prevalent kind of diabetes and is caused by a combination of different genetic and environmental factors, whereas rare phenotype monogenic diabetes is caused by a single gene mutation. Monogenic diabetes includes Neonatal diabetes mellitus and Maturity-onset diabetes of the young. The majority of our current knowledge about the pathogenesis of diabetes stems from studies done on animal models. However, the genetic difference between these creatures and humans makes it difficult to mimic human clinical pathophysiology, limiting their value in modeling key aspects of human disease. Human pluripotent stem cell technologies combined with genome editing techniques have been shown to be better alternatives for creating in vitro models that can provide crucial knowledge about disease etiology. This review paper addresses genome editing and human pluripotent stem cell technologies for in vitro monogenic diabetes modeling.
... One example searched for sequences that differed by at least 3 bp from the dTALE-binding sequence and were absent from all promoter sequences (up to 2 kbp upstream of the ATG) in the human genome (Garg et al., 2012). However, three mismatches may not be sufficient to prevent binding of the dTALE because there is growing evidence that the impact of mismatches on the ability of RVDs to bind cognate bases depends on the combined effects of RVD-type, position within the EBE, overall RVD-composition, and the number of repeats (Juillerat et al., 2015;Meckler et al., 2013;Miller et al., 2015;Rinaldi et al., 2017;Rogers et al., 2015;Streubel et al., 2012). With this in mind, position-dependent base preferences for canonical RVDs (those with amino acid variants HD, NI, NG, or NN) have been evaluated and have been used to rate the impact of specific RVD-base mismatches in the context of the repeat array (Erkes et al., 2019;Miller et al., 2015). ...
In biological discovery and engineering research there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue‐types and/or at specific times often precludes co‐expression of multiple transgenes in precisely‐controlled developmental contexts. Here we developed a system for use in rice that comprises synthetic designer transcription activator‐like effectors (dTALEs) and cognate synthetic TALE‐activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems – dTALE1‐STAP1 and dTALE2‐STAP2 – can activate STAP‐driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell‐types, requiring empirical evaluation in each case. dTALE expression leads to off‐target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE‐STAP system thus provides a powerful approach to fine‐tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.
... In a colinear manner, amino acid 13 from successive repeats along the TALE central domain sequence recognizes a cognate nucleobase along the EBE strand. Subsequent to its discovery [17,18], the RVD-nucleotide association code has been relatively well characterized and prominent RVDs, such as NI, NG, HD, and NN, were shown to recognize A, T, C, and G/A, respectively [19]. As a general rule and with the exception of RipTALs [20], EBEs start with a T that is not recognized by a canonical repeat but rather by a structurally related helical bundle or a cryptic repeat located before the central domain [21][22][23]. ...
Xanthomonas oryzae pv. oryzae (Xoo) strains that cause bacterial leaf blight (BLB) limit rice (Oryza sativa) production and require breeding more resistant varieties. Transcription activator-like effectors (TALEs) activate transcription to promote leaf colonization by binding to specific plant host DNA sequences termed effector binding elements (EBEs). Xoo major TALEs universally target susceptibility genes of the SWEET transporter family. TALE-unresponsive alleles of clade III OsSWEET susceptibility gene promoter created with genome editing confer broad resistance on Asian Xoo strains. African Xoo strains rely primarily on the major TALE TalC, which targets OsSWEET14. Although the virulence of a talC mutant strain is severely impaired, abrogating OsSWEET14 induction with genome editing does not confer equivalent resistance on African Xoo. To address this contradiction, we postulated the existence of a TalC target susceptibility gene redundant with OsSWEET14. Bioinformatics analysis identified a rice locus named ATAC composed of the INCREASED LEAF INCLINATION 2 (ILI2) gene and a putative lncRNA that are shown to be bidirectionally upregulated in a TalC-dependent fashion. Gain-of-function approaches with designer TALEs inducing ATAC sequences did not complement the virulence of a Xoo strain defective for SWEET gene activation. While editing the TalC EBE at the ATAC loci compromised TalC-mediated induction, multiplex edited lines with mutations at the OsSWEET14 and ATAC loci remained essentially susceptible to African Xoo strains. Overall, this work indicates that ATAC is a probable TalC off-target locus but nonetheless documents the first example of divergent transcription activation by a native TALE during infection.
... For ND5.2-DdCBEs containing DddA6 or DddA11, we observed fewer than four SNVs with more than 1% frequency-far lower than those observed in ATP8-DdCBE containing the same DddA6 or DddA11 (compare Extended Data Fig. 6b,c to 6e,f). We hypothesize that TALE repeats that bind promiscuously to multiple DNA bases are more likely to result in higher off-target editing when fused to the evolved DddA variants 31,32 . ...
The all-protein cytosine base editor DdCBE uses TALE proteins and a double-stranded DNA-specific cytidine deaminase (DddA) to mediate targeted C•G-to-T•A editing. To improve editing efficiency and overcome the strict TC sequence-context constraint of DddA, we used phage-assisted non-continuous and continuous evolution to evolve DddA variants with improved activity and expanded targeting scope. Compared to canonical DdCBEs, base editors with evolved DddA6 improved mitochondrial DNA (mtDNA) editing efficiencies at TC by 3.3-fold on average. DdCBEs containing evolved DddA11 offered a broadened HC (H = A, C or T) sequence compatibility for both mitochondrial and nuclear base editing, increasing average editing efficiencies at AC and CC targets from less than 10% for canonical DdCBE to 15–30% and up to 50% in cell populations sorted to express both halves of DdCBE. We used these evolved DdCBEs to efficiently install disease-associated mtDNA mutations in human cells at non-TC target sites. DddA6 and DddA11 substantially increase the effectiveness and applicability of all-protein base editing.
... Repeats with 35 aa often carry an additional proline at position 33 of the repeat, but other amino acid changes can occur as well. Despite the amino acid differences of those variants, they confer a normal DNA-binding mode (23,38,39). However, rare repeat variants with more profound differences in length and effect have been found in nature as well. ...
Transcription activator-like effectors (TALEs) are bacterial proteins with a programmable DNA-binding domain, which turned them into exceptional tools for biotechnology. TALEs contain a central array of consecutive 34 amino acid long repeats to bind DNA in a simple one-repeat-to-one-nucleotide manner. However, a few naturally occurring aberrant repeat variants break this strict binding mechanism, allowing for the recognition of an additional sequence with a −1 nucleotide frameshift. The limits and implications of this extended TALE binding mode are largely unexplored. Here, we analyse the complete diversity of natural and artificially engineered aberrant repeats for their impact on the DNA binding of TALEs. Surprisingly, TALEs with several aberrant repeats can loop out multiple repeats simultaneously without losing DNA-binding capacity. We also characterized members of the only natural TALE class harbouring two aberrant repeats and confirmed that their target is the major virulence factor OsSWEET13 from rice. In an aberrant TALE repeat, the position and nature of the amino acid sequence strongly influence its function. We explored the tolerance of TALE repeats towards alterations further and demonstrate that inserts as large as GFP can be tolerated without disrupting DNA binding. This illustrates the extraordinary DNA-binding capacity of TALEs and opens new uses in biotechnology.
... The interaction between an E gene and a TALE is a gene-for-gene relationship. TALEs have several well-characterized domains, which are conserved in the family and are essential for inducing the expression of their target genes [32][33][34]. A typical TALE has type III secretion and translocation signals in the N-terminal to direct the effector protein to enter host cells via the bacterial type III secretion system (T3SS). ...
Executor (E) genes comprise a new type of plant resistance (R) genes, identified from host–Xanthomonas interactions. The Xanthomonas-secreted transcription activation-like effectors (TALEs) usually function as major virulence factors, which activate the expression of the so-called “susceptibility” (S) genes for disease development. This activation is achieved via the binding of the TALEs to the effector-binding element (EBE) in the S gene promoter. However, host plants have evolved EBEs in the promoters of some otherwise silent R genes, whose expression directly causes a host cell death that is characterized by a hypersensitive response (HR). Such R genes are called E genes because they trap the pathogen TALEs in order to activate expression, and the resulting HR prevents pathogen growth and disease development. Currently, deploying E gene resistance is becoming a major component in disease resistance breeding, especially for rice bacterial blight resistance. Currently, the biochemical mechanisms, or the working pathways of the E proteins, are still fuzzy. There is no significant nucleotide sequence homology among E genes, although E proteins share some structural motifs that are probably associated with the signal transduction in the effector-triggered immunity. Here, we summarize the current knowledge regarding TALE-type avirulence proteins, E gene activation, the E protein structural traits, and the classification of E genes, in order to sharpen our understanding of the plant E genes.
... As described in the previous section, we extended the previously trained PrediTALE model by adding parameters for the specificity to bind to '5mC' to incorporate methylation information into the TALE target prediction of EpiTALE. The former training [19] included pairs of TALEs and their putative target boxes from different experiments [37][38][39][40][41]. ...
Background
The yield of many crop plants can be substantially reduced by plant-pathogenic Xanthomonas bacteria. The infection strategy of many Xanthomonas strains is based on transcription activator-like effectors (TALEs), which are secreted into the host cells and act as transcriptional activators of plant genes that are beneficial for the bacteria.The modular DNA binding domain of TALEs contains tandem repeats, each comprising two hyper-variable amino acids. These repeat-variable diresidues (RVDs) bind to their target box and determine the specificity of a TALE.All available tools for the prediction of TALE targets within the host plant suffer from many false positives. In this paper we propose a strategy to improve prediction accuracy by considering the epigenetic state of the host plant genome in the region of the target box.
Results
To this end, we extend our previously published tool PrediTALE by considering two epigenetic features: (i) chromatin accessibility of potentially bound regions and (ii) DNA methylation of cytosines within target boxes. Here, we determine the epigenetic features from publicly available DNase-seq, ATAC-seq, and WGBS data in rice.We benchmark the utility of both epigenetic features separately and in combination, deriving ground-truth from RNA-seq data of infections studies in rice. We find an improvement for each individual epigenetic feature, but especially the combination of both.Having established an advantage in TALE target predicting considering epigenetic features, we use these data for promoterome and genome-wide scans by our new tool EpiTALE, leading to several novel putative virulence targets.
Conclusions
Our results suggest that it would be worthwhile to collect condition-specific chromatin accessibility data and methylation information when studying putative virulence targets of Xanthomonas TALEs.
... Unalike zinc fingers, that verify DNA triplets, each TALE repeat recognizes only a single bp, with little to no target site overlap from adjacent domains (Mak et al., 2012). The most generally used RVDs for assembling synthetic TALE arrays are: NI for adenine, HD for cytosine, NG for thymine, and NN or HN for guanine or adenine (Streubel et al., 2012). TALE DNA-binding domains can be composed using a different method, with the most straightforward approach being Golden Gate assembly (Cermak et al., 2011). ...
Genome engineering is one of the worldwide fast growing field of biotechnology which involves designed programmable DNA-binding nucleases such as homing endonucleases, zinc finger nucleases (ZFNs), transcriptions activator like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (CRISPR-associated 9) nucleases. These technologies utilize manipulated nucleases which are the complex of sequence-specific DNA binding domains and nonspecific DNA cleavage modules. CRISPR)/Cas9 technology lets scientists accurately cut and paste genes into DNA which can be applied to edit the individual gene or even entire chromosomes from an organism at any point in its development, become a magical tool due to its simplicity. Here we review the four basic pieces of information on the genome editing technologies with their reliability and discuss the applications and their therapeutic potential as well as future prospects.
... As described in the previous section, we extended the previously trained PrediTALE model by adding parameters for the specificity to bind to '5mC' to incorporate methylation information into the TALE target prediction of EpiTALE. The former training (13) included pairs of TALEs and their putative target boxes from different experiments (37)(38)(39)(40)(41). A thorough study by Zhang et al. tested all theoretically possible combinations of RVDs to bind to 5methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), cytosine and thymine (16). ...
The yield of many crop plants can be substantially reduced by plant-pathogenic Xanthomonas bacteria. The infection strategy of many Xanthomonas strains is based on transcription activator-like effectors (TALEs), which are secreted into the host cells and act as transcriptional activators of plant genes that are beneficial for the bacteria.
The modular DNA binding domain of TALEs contains tandem repeats, each comprising two hyper-variable amino acids. These repeat-variable diresidues (RVDs) bind to a continuous DNA stretch (a target box) and determine the specificity of a TALE. All available tools for the prediction of TALE targets within the host plant suffer from many false positives. In this paper we propose a strategy to improve prediction accuracy by considering the epigenetic state of the host plant genome in the region of the target box. To this end, we extend our previously published tool PrediTALE by two epigenetic features: (i) We allow for filtering target boxes according to chromatin accessibility and (ii) we allow for considering the methylation state of cytosines within the target box during prediction, since DNA methylation may affect the binding specificity of RVDs. Here, we determine the epigenetic features from publicly available DNase-seq, ATAC-seq, and WGBS-seq data in rice.
We benchmark the utility of both epigenetic features separately and in combination, deriving ground-truth from RNA-seq infections studies in rice. We find an improvement for each individual epigenetic feature, but especially the combination of both. Having established an advantage in TALE target predicting considering epigenetic features, we use these data for promoterome and genome-wide scans by our new tool EpiTALE, leading to several novel putative virulence targets.
Our results suggest that it would be worthwhile to collect condition-specific chromatin accessibility data and methylation information when studying putative virulence targets of Xan-thomonas TALEs.
... The development of genome editing technology over the last 20 years has employed several types of nucleases (Boch et al. 2009;Christian et al. 2010;Durai et al. 2005;Joung and Sander 2013;Kim et al. 1996;Paques and Duchateau 2007;Smith et al. 2006;Streubel et al. 2012), but CRISPR/Cas9 has revolutionized genome editing because of its simplicity, versatility, efficiency, and specificity (Gil-Humanes et al. 2017;Li et al. 2016;Liang et al. 2017;Shan et al. 2014;Yin et al. 2017;Zhang et al. 2016). CRISPR/Cas9 has been used successfully in numerous organisms, including several plant species (Chang et al. 2016;Gao et al. 2015;Gerasimova et al. 2017;Jacobs et al. 2015;Jiang et al. 2013Jiang et al. , 2014Lowder et al. 2015;Michno et al. 2015;Yin et al. 2015;Zhang et al. 2016). ...
The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.
... Thus, each monomer or TALE repeat recognizes a single base pair in a contiguous DNA sequence through its corresponding RVDs and establishes a simple 1:1 code for the protein-to-DNA interaction. The binding behaviour and the binding affnity of various RVDs with their corresponding base pair are clearly mentioned in Table 3.1 (Boch et al., 2009;Moscou and Bogdanove, 2009;Miller et al., 2011;Streubel et al., 2012;Meckler et al., 2013). ...
... This allowed the Paired Target Finder feature of TAL effector-nucleotide targeter (TALE-NT) 75 to sum up the relative score of each RVD-nucleotide association using the frequency matrix for potential target sites. The search tool TALENoffer 76 further incorporates the contributions of different RVDs to TALEN cutting activity 77 . ...
Genome editing using programmable nucleases is revolutionizing life science and medicine. Off-target editing by these nucleases remains a considerable concern, especially in therapeutic applications. Here we review tools developed for identifying potential off-target editing sites and compare the ability of these tools to properly analyze off-target effects. Recent advances in both in silico and experimental tools for off-target analysis have generated remarkably concordant results for sites with high off-target editing activity. However, no single tool is able to accurately predict low-frequency off-target editing, presenting a bottleneck in therapeutic genome editing, because even a small number of cells with off-target editing can be detrimental. Therefore, we recommend that at least one in silico tool and one experimental tool should be used together to identify potential off-target sites, and amplicon-based next-generation sequencing (NGS) should be used as the gold standard assay for assessing the true off-target effects at these candidate sites. Future work to improve off-target analysis includes expanding the true off-target editing dataset to evaluate new experimental techniques and to train machine learning algorithms; performing analysis using the particular genome of the cells in question rather than the reference genome; and applying novel NGS techniques to improve the sensitivity of amplicon-based off-target editing quantification.
... The DNA-binding domain consists of monomers of the tandem repeats of about 34 amino acid residues; two amino acids at the position of 12 and 13 being highly variable are known as the repeat variable di-residue (RVD). The monomers are responsible for the target sequence of construct to undergo binding with specific nucleotide residues (Boch et al., 2009;Cong et al., 2012;Moscou and Bogdanove, 2009;Streubel et al., 2012). TALENs show similarity with the ZFNs, as they also contain a non-specific Fok1 domain. ...
The versatility of Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) genome editing tool ushered biologists into an exciting era of editing genomes with great efficiency and at a pace that was never imagined before. Though the CRISPR/Cas genome editing was developed after Zinc Finger Nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs), it is more popular and successful than these genome editing systems. The advent of targetable nucleases such as Cas9 has enabled manipulation of genomes in an accurate and precise manner. The CRISPR/Cas system of editing plant genomes has technical and economical advantages over conventional breeding methods. It has led to the development of traits within plant genomes that fulfill the needs of mankind. Advent of innovative procedures have paved the way for effective and efficient genome editing that has revolutionized genetic aspects and meets the safety regulations toward development of crops. The present review highlights the critical aspects of employing CRISPR/Cas for editing plant genomes in comparison with previously known editing approaches, such as ZFNs and TALENs. The study includes descriptive information on the approaches, procedural programs and applications in editing plant genomes for improving traits such as crop yield, resistance against emerging pathogens, abiotic stresses and herbicide tolerance thereof in the present-day world.
... The customizable binding domain of TALE DNA comprises of indubitable identical repeat arrays in tandem that could spot any furnished sequence based on simple RVD (repeat variable di-residue) code for nucleotide recognition . It is reported that in RVD the initial amino acid residue (N or H) is important for the offset of spatial conformation even if it does not straightaway linearly attach to a nucleotide, since the other alternative amino acid residue binds to a nucleotide through H-N bases or using van der Waals forces (Streubel et al., 2012). ...
The formulation of plants with ameliorated high-quality attributes particularly disease impedance, protracted shelf-life and drought withstanding using prevailing breeding comes out to be immensely sluggish. Nonetheless, the consumption is day by day increasing by virtue of humongous growth in global populace and transformation in consumption patterns. In this connection, genetically modified (GM) crops provide viable basis for safeguarding food availability for burgeoning global community. Since their introduction from 1996, a 100-fold increase in GM crop production has been observed and it is providing substantial multiple benefits to farmers. GM crops lend us ecstatic circumstances to upsurge food and feed yielding skilfully by causing plant between superior yield and superior nutritional yields by using various gene-editing methods. Genome editing of plants and genetic transformation have played an important role in GM crop production through beneficial novel genes insertion or silencing. However, transfer and assimilation of unfamiliar offshore genes at definitive pre-contemplated locale preclude umpteen intricacies linked with the extant gene transfer mechanisms. This chapter aims to provide information on advanced gene targeting techniques used
in plants by employing zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindrome repeats in recent years.
... Meganucleases recognize conserved sequences of 12-42 nt, while ZNFs consist of two modules of tandem repeat DNA-binding domains flanking the FokI nuclease catalytic domain, where a binding domain recognizes a unique nucleotide triplet, and each module recognizes up to 24 nt. In contrast, TALENs also comprise two modules of tandem repeat DNA-binding motifs flanking a FokI motif, but each binding domain recognizes only one nucleotide (Streubel et al., 2012). In the last 10 years, CRISPR/Cas9 or optimized nucleases (e.g., CRISPR/Cpf1 or CRISPR/Csm1) have been successfully used in plant genome editing (Osakabe et al., 2016;. ...
Climate change and the exploration of new areas of cultivation have impacted the yields of several economically important crops worldwide. Both conventional plant breeding based on planned crosses between parents with specific traits and genetic engineering to develop new biotechnological tools (NBTs) have allowed the development of elite cultivars with new features of agronomic interest. The use of these NBTs in the search for agricultural solutions has gained prominence in recent years due to their rapid generation of elite cultivars that meet the needs of crop producers, and the efficiency of these NBTs is closely related to the optimization or best use of their elements. Currently, several genetic engineering techniques are used in synthetic biotechnology to successfully improve desirable traits or remove undesirable traits in crops. However, the features, drawbacks, and advantages of each technique are still not well understood, and thus, these methods have not been fully exploited. Here, we provide a brief overview of the plant genetic engineering platforms that have been used for proof of concept and agronomic trait improvement, review the major elements and processes of synthetic biotechnology, and, finally, present the major NBTs used to improve agronomic traits in socioeconomically important crops.
TALEs (transcription activator-like effectors) in plant-pathogenic Xanthomonas bacteria activate expression of plant genes and support infection or cause a resistance response. PthA4AT is a TALE with a particularly short DNA-binding domain harbouring only 7.5-repeats which triggers cell death in Nicotiana benthamiana; however, the genetic basis for this remains unknown. To identify possible target genes of PthA4AT that mediate cell death in N. benthamiana, we exploited the modularity of TALEs to stepwise enhance their specificity and reduce potential target sites. Substitutions of individual repeats suggested that PthA4AT-dependent cell death is sequence-specific. Stepwise addition of repeats to the C-terminal or N-terminal end of the repeat region narrowed the sequence requirements in promoters of target genes. Transcriptome profiling and in silico target prediction allowed the isolation of two cell death-inducer genes, which encode a patatin-like protein and a bifunctional monodehydroascorbate reductase/carbonic anhydrase protein. These two proteins are not linked to known TALE-dependent resistance genes. Our results show that the aberrant expression of different endogenous plant genes can cause a cell death reaction, which supports the hypothesis that TALE-dependent executor resistance genes can originate from various plant processes. Our strategy further demonstrates the use of TALEs to scan genomes for genes triggering cell death and other relevant phenotypes.
Precise genome-editing platforms are versatile tools for generating specific, site-directed DNA insertions, deletions, and substitutions. The continuous enhancement of these tools has led to a revolution in the life sciences, which promises to deliver novel therapies for genetic disease. Precise genome-editing can be traced back to the 1950s with the discovery of DNA’s double-helix and, after 70 years of development, has evolved from crude in vitro applications to a wide range of sophisticated capabilities, including in vivo applications. Nonetheless, precise genome-editing faces constraints such as modest efficiency, delivery challenges, and off-target effects. In this review, we explore precise genome-editing, with a focus on introduction of the landmark events in its history, various platforms, delivery systems, and applications. First, we discuss the landmark events in the history of precise genome-editing. Second, we describe the current state of precise genome-editing strategies and explain how these techniques offer unprecedented precision and versatility for modifying the human genome. Third, we introduce the current delivery systems used to deploy precise genome-editing components through DNA, RNA, and RNPs. Finally, we summarize the current applications of precise genome-editing in labeling endogenous genes, screening genetic variants, molecular recording, generating disease models, and gene therapy, including ex vivo therapy and in vivo therapy, and discuss potential future advances.
Transcription activator-like effector nucleases (TALENs) is a targeted genome editing approach used to modify any sequence of interest in living cells or organisms. TALENs rely on engineered nucleases and foster systematic, unbiased genome screening. These synthetic proteins are composed of sequence-specific DNA-binding domains, recognize the target site on the basis of DNA protein interaction, and cleave at specific sites to make effective genes nonfunctional, especially in cancer. Therefore, the development of this technology might provide therapeutic avenues in genomic aberrated cancers. Although the approach to the use TALENs in cancer management and treatment is long overdue, it needs more investigations along with adaptations. Innovative adaptations in this technique would increase the specificity and efficiency of the technique to facilitate gene therapy and cancer management. This chapter will highlight the molecular prospects of transcription activator-like effector nucleases (TALENs) in cancer management, challenges in execution, and adaptations for future investigations.
Repeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications.
Genome/gene-editing (GE) techniques, characterized by a low technological barrier, high efficiency, and broad application among organisms, are now being employed not only in medical science but also in agriculture/veterinary science. Different engineered CRISPR/Cas9s have been identified to expand the application of this technology. In pig production, GE is a precise new breeding technology (NBT), and promising outcomes in improving economic traits, such as growth, lean or healthy meat production, animal welfare, and disease resistance, have already been documented and reviewed. These promising achievements in porcine gene editing, including the Myostatin gene knockout (KO) in indigenous breeds to improve lean meat production, the uncoupling protein 1 (UCP1) gene knock-in to enhance piglet thermogenesis and survival under cold stress, the generation of GGTA1 and CMAH gene double KO pigs to produce healthy red meat, and the KO or deletion of exon 7 of the CD163 gene to confer resistance to porcine reproductive and respiratory syndrome virus (PRRSV) infection, are described in the present article. Other related approaches for such purposes are also discussed. The current trend of global regulations or legislation for GE organisms is that they are exempted from classification as genetically modified organisms (GMOs) if no exogenes are integrated into the genome, according to product-based and not process-based methods. Moreover, an updated case study in the EU showed that current GMO legislation is not fit for purpose in term of NBTs, which contribute to the objectives of the EU's Green Deal and biodiversity strategies and even meet the United Nations' sustainable development goals for a more resilient and sustainable agri-food system. The GE pigs generated via NBT will be exempted from classification as GMOs, and their global valorization and commercialization can be foreseen.
Accurate assessment of plant symptoms plays a key role for measuring the impact of pathogens during plant-pathogen interaction. Common bacterial blight caused by Xanthomonas phaseoli pv. phaseoli and Xanthomonas citri pv. fuscans (Xpp-Xcf) is a major threat to common bean. The pathogenicity of these bacteria is variable among strains, and depends mainly on a type III secretion system and associated type III effectors such as transcription activator-like effectors (TALEs). Because the impact of a single gene is often small and difficult to detect, a discriminating methodology is required to distinguish the slight phenotype changes induced during the progression of the disease. Here, we compared two different inoculation and symptom assessment methods for their ability to distinguish two tal mutants from their corresponding wild-type strains. Interestingly, rub-inoculation of the first leaves combined with symptom assessment by machine learning-based imaging allowed significant distinction between wild-type and mutant strains. By contrast, dip-inoculation of first trifoliate leaves combined with chlorophyll fluorescence imaging did not differentiate the strains. Furthermore, the new method developed here led to the miniaturization of pathogenicity tests and significant time savings.
Genome editing enables the study of gene functions and the construction of biological systems useful to society. The discovery that double‐stranded breaks in DNA could be used to mutate DNA sequences with non‐homologous end joining or integrate foreign DNA at high rates with homologous recombination gave rise to endonuclease‐based genome editing technologies that allowed for unprecedented precision and control. The development of meganucleases, zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) revolutionized the field to the point where ever more sophisticated genome editing techniques are becoming accessible routine procedures applicable for an ever‐increasing number of organisms. This chapter seeks to describe the current technologies for genome editing and their application in industrially relevant eukaryotic organisms.
Horizontal gene transfer is of major evolutionary importance as it allows for the redistribution of phenotypically important genes among lineages. Such genes with essential functions include those involved in resistance to antimicrobial compounds and virulence factors in pathogenic bacteria. Understanding gene turnover at microevolutionary scales is critical to assess the pace of this evolutionary process. Here we characterized and quantified gene turnover for the epidemic lineage of a bacterial plant pathogen of major agricultural importance worldwide. Relying on a dense geographic sampling spanning 39 years of evolution, we estimated both the dynamics of single nucleotide polymorphism accumulation and gene content turnover. We identified extensive gene content variation among lineages even at the smallest phylogenetic and geographic scales. Gene turnover rate exceeded nucleotide substitution rate by three orders of magnitude. Accessory genes were found preferentially located on plasmids, but we identified a highly plastic chromosomal region hosting ecologically important genes such as transcription activator‐like effectors. Whereas most changes in the gene content are probably transient, the rapid spread of a mobile element conferring resistance to copper compounds widely used for the management of plant bacterial pathogens illustrates how some accessory genes can become ubiquitous within a population over short time‐frames.
Most plant agronomic traits are quantitatively inherited. Identification of quantitative trait loci (QTL) is a challenging target for most scientists and crop breeders as large-scale genotyping is difficult. Molecular marker technology has continuously evolved from hybridization-based technology to PCR-based technology, and finally, sequencing-based high-throughput single-nucleotide polymorphisms (SNPs). High-throughput sequencing technologies can provide strategies for sequence-based SNP genotyping. Here we describe the SLAF-seq that can be applied as the SNP genotyping approach. The high-throughput SNP genotyping methods will prove useful for the construction of high-density genetic maps and identification of QTLs for their deployment in plant breeding and facilitate genome-wide selection (GWS) and genome-wide association studies (GWAS).
Phenotyping root systems provide essential information for plant breeding, particularly aiming for better abiotic stress resistance. Rhizobox systems provide a field-near growth environment for in situ imaging of root systems in soil. A protocol for RGB and hyperspectral imaging of rhizobox-grown plants is presented that enables gathering of root structural (morphology, architecture) as well as functional (water content, decomposition) information. The protocol exemplifies the setup of a root phenotyping platform combining low-cost RGB with advanced short-wave infrared hyperspectral imaging. For both types of imaging approach, the essential steps of an image analysis pipeline are provided to retrieve biological information on breeding-relevant traits from the imaging datasets.
Transcription activator-like effector (TALE) is a DNA-binding domain that can be paired with a nuclease to create DNA double-strand breaks, or with an effector protein to alter gene transcription. The ability to precisely alter plant genomes and transcriptomes has provided many insights into gene function and has recently been utilized for crop improvement. Easy design and construction of TALE make the tool more accessible to a variety of researchers. Here, we describe two TALE-based systems: transcription activator-like effector nucleases (TALEN), for creating targeted mutations in a gene of interest, and multiplex TALE activation (mTALE-Act), for activating one or a few genes of interest at the transcription level. Assembly of these tools is based on Golden Gate cloning and Gateway recombination, which are cost-effective and streamlined cloning methods.
Multiparental populations are located midway between association mapping that relies on germplasm collections and classic linkage analysis, based upon biparental populations. They provide several key advantages such as the possibility to include a higher number of alleles and increased level of recombination with respect to biparental populations, and more equilibrated allelic frequencies than association mapping panels. Moreover, in these populations new allele’s combinations arise from recombination that may reveal transgressive phenotypes and make them a useful pre-breeding material. Here we describe the strategies for working with multiparental populations, focusing on nested association mapping populations (NAM) and multiparent advanced generation intercross populations (MAGIC). We provide details from the selection of founders, population development, and characterization to the statistical methods for genetic mapping and quantitative trait detection.
The global climate is changing, resulting in significant economic losses worldwide. It is thus necessary to speed up the plant selection process, especially for complex traits such as biotic and abiotic stresses. Nowadays, genomic selection (GS) is paving new ways to boost plant breeding, facilitating the rapid selection of superior genotypes based on the genomic estimated breeding value (GEBV). GEBVs consider all markers positioned throughout the genome, including those with minor effects. Indeed, although the effect of each marker may be very small, a large number of genome-wide markers retrieved by high-throughput genotyping (HTG) systems (mainly genotyping-by-sequencing, GBS) have the potential to explain all the genetic variance for a particular trait under selection. Although several workflows for GBS and GS data have been described, it is still hard for researchers without a bioinformatics background to carry out these analyses. This chapter has outlined some of the recently available bioinformatics resources that enable researchers to establish GBS applications for GS analysis in laboratories. Moreover, we provide useful scripts that could be used for this purpose and a description of key factors that need to be considered in these approaches.
This volume describes breeding methods for the development of biparental and multiparental mapping populations. Chapters detail lab protocols for high-throughput isolation of nucleic acids and metabolites, high performing genotyping approaches, mapping strategies for QTLs, mutation identifications, computational, bioinformatic pipelines, tissue culture-based and transformation methods for androgenesis, ploidy modification, and RNA interference. Additional chapters highlight recent developed genome editing protocols including CRISPR and TALEN methods and methodologies for in-field/in-soil plant phenotyping. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, Crop Breeding: Genetic Improvement Methods aims to ensure successful results in the further study of this vital field.
The Golgi is essential for glycosylation of newly synthesised proteins including almost all cell-surface and extracellular matrix proteoglycans. Giantin, encoded by the golgb1 gene, is a member of the golgin family of proteins that reside within the Golgi stack but its function remains elusive. Loss-of-function of giantin in rats causes osteochondrodysplasia; knockout mice show milder defects, notably a cleft palate. In vitro, giantin has been implicated in Golgi organization, biosynthetic trafficking, and ciliogenesis. Here we show that loss-of-function of giantin in zebrafish, using either morpholino or knockout techniques, causes defects in cilia function. Giantin morphants have fewer cilia in the neural tube and those remaining are longer. Mutants have the same number of cilia in the neural tube but these cilia are also elongated. Scanning electron microscopy shows that loss of giantin results in an accumulation of material at the ciliary tip, consistent with a loss-of-function of retrograde intraflagellar transport. Mutants show milder defects than morphants consistent with adaptation to loss of giantin.
Summary statement
Loss of giantin following either morpholino injection or genome editing in zebrafish results in defects in ciliogenesis.
This chapter reviews the principle of the transgenic fish technology as well as its application in basic research and biotechnology applications. It discusses the characterization of transgenic fish, including identifying transgenic individuals, characterizing transgene integration, and breeding homozygous transgenic fish. In recent years, transgenic fish technology has made major contributions in the areas of the study of vertebrate development, the analysis of promoter/enhancer elements of genes, genome editing, and the development of human disease models. Transgenic fish technology can facilitate the genetic selection process by directly modifying the undesirable genetic traits that confer vulnerability of fish to pathogens or introducing specific genes that are related to disease resistance into fish. The introduced transgenes can be fish‐originated or characterized genes from other species. The application of transgenic fish technology to produce fish with beneficial traits, such as environmental biomonitors and genetically modified food, is on the rise.
Abiotic stresses are the major prevailing forms of environmental contaminants that result in harmful effects in plants and cause foremost environmental problems globally. A general effect of abiotic stress is the extreme accumulation of reactive oxygen species that can cause lipid peroxidation, oxidation of protein, inactivation of enzymes, DNA damage and interact with other imperative constituents of plant cells. Higher plants have evolved an intricate antioxidant defence system to scavenge reactive oxygen species during abiotic stress conditions. Genome editing approaches propose numerous applications in the improvement of crops towards abiotic stress tolerance, and produce quality improvement. Utilizing transgenic approaches, functional validation of several target genes engage in different processes, viz signalling, transcription, homeostasis, antioxidant defence for enhanced abiotic stress resistance has been employed in different plants including Brassicaceae crop plants. This chapter provides an inclusive outline to illustrate the interest of researchers for a better understanding of genome editing advancements in relation to abiotic stress tolerance in different Brassicaceae crops.
Medicinal plants have been used for therapeutic purposes and in food and other industries for a long time. The biotechnology based breeding methods (BBBMs) have been applied to these valuable plants. Genetics and biotechnology can help to improve medicinal plants faster through assessment of the genetic diversity, conservation, proliferation, and overproduction. Genetic and metabolic engineering techniques have enabled manipulation by modifying the genes that play a key role in the biosynthetic pathway and production of specific plant secondary metabolites. For functional improvement of crops as well as for studying basic molecular biology in plants, plant genetic transformation has become an essential research tool. In order to increase the efficiency of transformation and to achieve stable expression of transgenes in plants various methodologies of plant transformation have been developed. Various genome-editing methods like sequence-specific nucleases of transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and clustered regularly interspaced short palindromic repeats-associated (Cas) can produce user-designed medicinal plants. Recently, Agrobacterium rhizogenes mediated transgenic hairy root system provides potential for introducing foreign genes along with the Ri plasmid into plant cells for increased production of secondary metabolites. These current targeted genome editing methods open new gates to medicinal plants to be introduced into appropriate industries.
Wrapped DNA
TAL effectors are proteins that bacterial pathogens inject into plant cells that bind to host DNA to activate expression of plant genes. The DNA-binding domain of TAL proteins is composed of tandem repeats within which a repeat-variable diresidue sequence confers nucleotide specificity. Deng et al. (p. 720 , published online 5 January) report the structure of the TAL effector dHax3, containing 11.5 repeats, in DNA-free and DNA-bound states, and Mak et al. (p. 716 , published online 5 January) report the structure of the PthXo1 TAL effector, containing 22 repeats, bound to its DNA target. Together, the structures reveal the conformational changes involved in DNA binding and provide the structural basis of DNA recognition.
DNA recognition by TAL effectors is mediated by tandem repeats, each 33 to 35 residues in length, that specify nucleotides
via unique repeat-variable diresidues (RVDs). The crystal structure of PthXo1 bound to its DNA target was determined by high-throughput
computational structure prediction and validated by heavy-atom derivatization. Each repeat forms a left-handed, two-helix
bundle that presents an RVD-containing loop to the DNA. The repeats self-associate to form a right-handed superhelix wrapped
around the DNA major groove. The first RVD residue forms a stabilizing contact with the protein backbone, while the second
makes a base-specific contact to the DNA sense strand. Two degenerate amino-terminal repeats also interact with the DNA. Containing
several RVDs and noncanonical associations, the structure illustrates the basis of TAL effector–DNA recognition.
The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator-like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.
Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.
TAL Order
Xanthomonas bacteria attack their plant hosts by delivering their own transcription-activator–like (TAL) proteins into the plant cell nucleus and alter the plant's gene regulation (see the Perspective by Voytas and Joung ). Moscou and Bogdanove (p. 1501 , published online 29 October: see the cover) and Boch et al. (p. 1509 , published online 29 October) have now discovered how the similar but not identical repeats in the TAL proteins encode the specificity needed for the proteins to find their targets. Each repeat is specific for one DNA base pair, a specificity encoded by hypervariable amino acid positions. Combining several repeats with different amino acids in the hypervariable positions allowed the production of new effectors that targeted new DNA sites.
Generating and applying new knowledge from the wealth of available genomic information is hindered, in part, by the difficulty of altering nucleotide sequences and expression of genes in living cells in a targeted fashion. Progress has been made in engineering DNA binding domains to direct proteins to particular sequences for mutagenesis or manipulation of transcription; however, achieving the requisite specificities has been challenging. Transcription activator-like (TAL) effectors of plant pathogenic bacteria contain a modular DNA binding domain that appears to overcome this challenge. Comprising tandem, polymorphic amino acid repeats that individually specify contiguous nucleotides in DNA, this domain is being deployed in DNA targeting for applications ranging from understanding gene function in model organisms to improving traits in crop plants to treating genetic disorders in people.
Proteins that can be tailored to bind desired DNA sequences are key tools for molecular biology. Previous studies suggested that DNA-binding specificity of transcription activator-like effectors (TALEs) from the bacterial genus Xanthomonas is defined by repeat-variable diresidues (RVDs) of tandem-arranged 34/35-amino acid repeat units. We have studied chimeras of two TALEs differing in RVDs and non-RVDs and found that, in contrast to the critical contributions by RVDs, non-RVDs had no major effect on the DNA-binding specificity of the chimeras. This finding suggests that one needs only to modify the RVDs to generate designer TALEs (dTALEs) to activate transcription of user-defined target genes. We used the scaffold of the TALE AvrBs3 and changed its RVDs to match either the tomato Bs4, the Arabidopsis EGL3, or the Arabidopsis KNAT1 promoter. All three dTALEs transcriptionally activated the desired promoters in a sequence-specific manner as mutations within the targeted DNA sequences abolished promoter activation. This study is unique in showing that chromosomal loci can be targeted specifically by dTALEs. We also engineered two AvrBs3 derivatives with four additional repeat units activating specifically either the pepper Bs3 or UPA20 promoter. Because AvrBs3 activates both promoters, our data show that addition of repeat units facilitates TALE-specificity fine-tuning. Finally, we demonstrate that the RVD NK mediates specific interaction with G nucleotides that thus far could not be targeted specifically by any known RVD type. In summary, our data demonstrate that the TALE scaffold can be tailored to target user-defined DNA sequences in whole genomes.
The pathogenicity of many bacteria depends on the injection of effector proteins via type III secretion into eukaryotic cells
in order to manipulate cellular processes. TAL (transcription activator–like) effectors from plant pathogenic Xanthomonas are important virulence factors that act as transcriptional activators in the plant cell nucleus, where they directly bind
to DNA via a central domain of tandem repeats. Here, we show how target DNA specificity of TAL effectors is encoded. Two hypervariable
amino acid residues in each repeat recognize one base pair in the target DNA. Recognition sequences of TAL effectors were
predicted and experimentally confirmed. The modular protein architecture enabled the construction of artificial effectors
with new specificities. Our study describes the functionality of a distinct type of DNA binding domain and allows the design
of DNA binding domains for biotechnology.
- L Defrancesco
- F Zhang