Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining.
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TLDR
ObLiGaRe is described, a new method for site-specific gene insertions that uses the efficient NHEJ pathway and acts independently of HR, and has enabled us to insert a 15-kb inducible gene expression cassette at a defined locus in human cell lines.Abstract:Â
Custom-designed nucleases (CDNs) greatly facilitate genetic engineering by generating a targeted DNA double-strand break (DSB) in the genome. Once a DSB is created, specific modifications can be introduced around the breakage site during its repair by two major DNA damage repair (DDR) mechanisms: the dominant but error-prone nonhomologous end joining (NHEJ) pathway, and the less-frequent but precise homologous recombination (HR) pathway. Here we describe ObLiGaRe, a new method for site-specific gene insertions that uses the efficient NHEJ pathway and acts independently of HR. This method is applicable with both zinc finger nucleases (ZFNs) and Tale nucleases (TALENs), and has enabled us to insert a 15-kb inducible gene expression cassette at a defined locus in human cell lines. In addition, our experiments have revealed the previously underestimated error-free nature of NHEJ and provided new tools to further characterize this pathway under physiological and pathological conditions.read more
Citations
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Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.
Bernd Zetsche,Jonathan S. Gootenberg,Omar O. Abudayyeh,Ian Slaymaker,Kira S. Makarova,Patrick Essletzbichler,Sara E. Volz,Julia Joung,John van der Oost,Aviv Regev,Aviv Regev,Eugene V. Koonin,Feng Zhang +12 more
TL;DR: In this paper, the authors characterized Cpf1, a putative class 2 CRISPR effector, which is a single RNA-guided endonuclease lacking tracrRNA and utilizes a T-rich protospacer-adjacent motif.
Journal ArticleDOI
ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering
TL;DR: A review of achievements made possible by site-specific nuclease technologies and applications of these reagents for genetic analysis and manipulation, including the therapeutic potential of ZFNs and TALENs, and future prospects for the field are discussed.
Journal ArticleDOI
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity
F. Ann Ran,Patrick D. Hsu,Chie Yu Lin,Jonathan S. Gootenberg,Silvana Konermann,Alexandro E. Trevino,David A. Scott,Azusa Inoue,Shogo Matoba,Yi Zhang,Feng Zhang +10 more
TL;DR: In this paper, an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks is described. But the approach is limited to mouse zygotes.
Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity
F. Ann Ran,Patrick D. Hsu,Chie Yu Lin,Jonathan S. Gootenberg,Silvana Konermann,Alexandro E. Trevino,David A. Scott,Azusa Inoue,Shogo Matoba,Yi Zhang,Feng Zhang +10 more
TL;DR: It is demonstrated that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency.
Journal ArticleDOI
Easy quantitative assessment of genome editing by sequence trace decomposition
TL;DR: TIDE, a method that requires only a pair of PCR reactions and two standard capillary sequencing runs to identify the major induced mutations in the projected editing site and accurately determines their frequency in a cell population, is presented.
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