The I-TevI Nuclease and Linker Domains Contribute to the Specificity of Monomeric TALENs

Kleinstiver, Benjamin P.; Wang, Li; Wolfs, Jason M.; Kolaczyk, Tomasz; McDowell, Brendon; Xu, Wei; Schild-Poulter, Caroline; Bogdanove, Adam J.; Edgell, David R.

doi:10.1534/g3.114.011445

Cited by 27 publications

(29 citation statements)

References 43 publications

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“…The I-TevI nuclease and linker domains have successfully been fused to four different DNA-binding architectures that are used in genome editing: zinc fingers (27), TALEs (29), meganucleases (30), and, as reported here, Cas9. An on-going debate in the genomeediting field centers on the ease of use (targeting range) versus specificity (off-target effects) of the various reagents, particularly for common laboratory manipulations of cell lines or model organisms.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies revealed that the length of the DNA spacer is crucial for I-TevI cleavage function, with lengths of 14-19 bp supporting activity in various chimeric contexts (27,29,30). We coexpressed TevCas9 with a C-terminal histidine tag and a gRNA targeting a site in the retinoic acid receptor alpha gene (RARA.233, numbered according to start of target site in the RARA cDNA) from the same plasmid in Escherichia coli.…”

Section: Significancementioning

confidence: 99%

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Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

Wolfs

Hamilton

Lant

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33-to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.CRISPR/Cas9 | genome editing | NHEJ | I-TevI homing endonuclease G enome editing with engineered nucleases has revolutionized the targeted manipulation of the genomes of organisms ranging from bacteria to mammals (1). Zinc finger nucleases (ZFNs) (2), transcription-like effector nucleases (TALENs) (3), MegaTALs (fusion of a LAGLIDADG homing endonuclease and TALE domain) (4-6), and nucleases based on the CRISPR-associated protein 9 (Cas9) all represent programmable genome-editing nucleases that have successfully been used to introduce targeted changes in genomes (7-11). One of the most common applications of genome-editing nucleases is gene knockouts that are performed in the absence of an exogenously added repair template (12). In the case of Cas9, the blunt DNA ends introduced at DNA cleavage are substrates for error-free repair by the classical nonhomologous endjoining repair (c-NHEJ) pathway (13), regenerating the target site for recleavage by the nuclease. This cycle of cleavage, ligation, and target site regeneration is perturbed when the double-strand break (DSB) is sufficiently modified by exonucleolytic processing by c-NHEJ, or by the alternative NHEJ pathway (alt-NHEJ), to prevent cleavage by the nuclease (14-18). Imprecise repair by either of the NHEJ pathways generates the characteristic spectrum of heterogeneous length insertions or deletions (indels) centered around the break site (19,20). The heterogeneous distribution of indels, and the fact that not all indels generate gene knoc...

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Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

Wolfs

Hamilton

Lant

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Engineering nicking enzymes in all kinds of the chimeric artificial nucleases also resulted in a substantial progress of this field [46][47][48][49][50][51][52]. Alternative nuclease domains have been applied in few cases in mainly monomeric chimera [53][54][55][56][57][58] and non-FokI-based zinc finger nucleases have also been developed by introducing mutations or small modifications into the zinc finger array, such as the exchange of the metal ion-binding cysteines to histidines [59][60][61]. Trials to establish controlled artificial nucleases through lightactivation [62,63], DNA binding [64], cold-shock technique [65], protein-protein interaction [66], DNA modification [67] or metal ion dependent DNA binding [68] were also executed.…”

Section: Current Artificial Nucleasesmentioning

confidence: 99%

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

Gyurcsik

2016

CPPS

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Artificial nucleases are designed for in vivo gene engineering, as the DNA cleavage performed at a specific target site enhances the effectiveness of the cell's DNA repair machinery. The therapeutic potential of the above phenomenon stems from the knowledge that (i) the shifted reading frame can be restored by non-homologous end-joining, or (ii) a DNA of erroneous sequence -causing a genetic disease -can be corrected by homologous recombination in the presence of a suitable DNA template. Besides the advantageous properties of the nowadays applied zinc finger nucleases, TALE nucleases and the CRISPR/Cas9 system, they possess a residual citotoxicity. This is related to offtarget cleavages, which could be prevented by the strict regulation of the enzymes. The studies on enzymes acting naturally in a controlled manner are beneficial to get better insight into their mechanism. Such enzymes or their appropriate domains may be the most promising alternatives to the presently applied ones. As an example, the DNA cleavage of the inactive HNH nuclease mutants is inducible in a multiple way. This property may be used for establishing a control mechanism and thus, in combination with specific DNA-binding domains they are good candidates for the catalytic site of artificial nucleases. Here we collect the results on the properties of the HNH nucleases that allow for their redesign into enzymes with possible therapeutic applications.

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“…Several groups have attempted to create a hybrid nuclease by fusing TALE DNA domains with monomeric meganucleases, such as I -TevI , I -AniI , and I -OnuI . The resulting nucleases, termed megaTAL or compact TALEN, have shown great potential with high specifi city and activity (Beurdeley et al 2013 ;Kleinstiver et al 2014 ;Boissel et al 2014 ). Because all the meganucleases fused in megaTALs are monomeric endonucleases, the new hybrid nuclease does not require dimerization to cleave target sites.…”

Section: Fusion He-talementioning

confidence: 99%

“…This approach helps expand the collection of meganucleases with distinct target site specifi city for genome engineering applications. The other strategy is to create a hybrid nuclease architecture by combining the TALE DNA binding domain, due to ease of engineering and versatility, with the monomeric nuclease domain from meganucleases (Beurdeley et al 2013 ;Kleinstiver et al 2014 ;Boissel et al 2014 ).…”

Section: Future Perspectivesmentioning

confidence: 99%

Advances in New Technology for Targeted Modification of Plant Genomes

Zhang

Puchta

Thomson³

2015

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The I-TevI Nuclease and Linker Domains Contribute to the Specificity of Monomeric TALENs

Cited by 27 publications

References 43 publications

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

Advances in New Technology for Targeted Modification of Plant Genomes

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