CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles

Lemos, Brenda; Kaplan, Adam C.; Bae, Ji Eun; Ferrazzoli, Alexander E.; Kuo, Jian-Long; Anand, R.; Waterman, David P.; Haber, James E.

doi:10.1073/pnas.1716855115

Cited by 173 publications

(141 citation statements)

References 51 publications

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“… Cas9-nuclease mechanism : consistent with previous reports ( 59 ), our findings suggest that Cas9 nuclease activity followed by NHEJ is the mechanism underlying the duplication of nucleotides at repaired sites (Figure 5A ). This finding is also supported by a recent study in budding yeast ( 67 ) and a biochemical analysis of Cas9 nuclease activity ( 68 ). Interestingly, the latter study also showed that the Cas9 RuvC domain is capable of exonuclease activity on the cleaved target which can lead to its bidirectional degradation ( 68 ).…”

Section: Discussionsupporting

confidence: 81%

Decoding non-random mutational signatures at Cas9 targeted sites

Taheri‐Ghahfarokhi

Taylor

Nitsch

et al. 2018

Nucleic Acids Research

115

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The mutation patterns at Cas9 targeted sites contain unique information regarding the nuclease activity and repair mechanisms in mammalian cells. However, analytical framework for extracting such information are lacking. Here, we present a novel computational platform called Rational InDel Meta-Analysis (RIMA) that enables an in-depth comprehensive analysis of Cas9-induced genetic alterations, especially InDels mutations. RIMA can be used to quantitate the contribution of classical microhomology-mediated end joining (c-MMEJ) pathway in the formation of mutations at Cas9 target sites. We used RIMA to compare mutational signatures at 15 independent Cas9 target sites in human A549 wildtype and A549-POLQ knockout cells to elucidate the role of DNA polymerase θ in c-MMEJ. Moreover, the single nucleotide insertions at the Cas9 target sites represent duplications of preceding nucleotides, suggesting that the flexibility of the Cas9 nuclease domains results in both blunt- and staggered-end cuts. Thymine at the fourth nucleotide before protospacer adjacent motif (PAM) results in a two-fold higher occurrence of single nucleotide InDels compared to guanine at the same position. This study provides a novel approach for the characterization of the Cas9 nucleases with improved accuracy in predicting genome editing outcomes and a potential strategy for homology-independent targeted genomic integration.

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Section: Discussionsupporting

confidence: 81%

Decoding non-random mutational signatures at Cas9 targeted sites

Taheri‐Ghahfarokhi

Taylor

Nitsch

et al. 2018

Nucleic Acids Research

115

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“…These indels, usually smaller than 50 bp, can be predicted from DNA sequence with high precision (5)(6)(7)(8). In particular, frequent occurrence of 1 bp insertions templated from around the cut site has been linked to Cas9-induced DSBs with 1 nt 5' overhang (9,10). The size of indels is typically increased by NHEJ inhibition, while inhibition of the core MMEJ proteins, such as Parp1 and Polθ, decreases them (3,(11)(12)(13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

Cas9-induced large deletions and small indels are controlled in a convergent fashion

Kosicki

Allen

Bradley

2020

Preprint

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Repair of Cas9-induced double-stranded breaks results primarily in formation of small indels, but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a novel library of clonal mouse embryonic stem cells bona fide deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a novel role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation.

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“…Furthermore, the protocol allows for better control of the cutting step, so that it can also be used, for example, to examine repair of DSBs in the same fashion that it is done in S. cerevisiae (Lemos et al, 2018). In this work, we have used the system to confirm that faithful NHEJ is a major repair mechanism of chromosomal DSBs in C. glabrata.…”

Section: Discussionmentioning

confidence: 99%

“…In yeasts, the system has been used, first and foremost, in S. cerevisiae, for genome engineering (DiCarlo et al, 2013) but also for the study of the repair of DSBs (Lemos et al, 2018) and for mating-type switching (Xie et al, 2018). Several versions of the CRISPR-Cas9 systems have also been designed for C. glabrata (Cen, Timmermans, Souffriau, Thevelein, & Van Dijck, 2017;Enkler, Richer, Marchand, Ferrandon, & Jossinet, 2016;Vyas et al, 2018), with success in obtaining repair events of both NHEJ and HR types.…”

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confidence: 99%

A new inducible CRISPR‐Cas9 system useful for genome editing and study of double‐strand break repair in Candida glabrata

Maroc

Fairhead

2019

Yeast

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In recent years, the CRISPR‐Cas9 system has proven extremely useful for genome editing in many species, including the model yeast Saccharomyces cerevisiae and other yeast species such as Candida glabrata. Inducible CRISPR‐Cas9 systems have the additional advantage of allowing to separate the transformation step of the organism by the CRISPR‐Cas9 system, from the cutting and repair steps. This has indeed been developed in S. cerevisiae, where most inducible expression systems rely on the GAL promoters. Unfortunately, C. glabrata is gal− and lacks the GAL genes, like many other yeast species. We report here the use of a vector expressing cas9 under the control of the MET3 promoter, with the guide RNA cloned into the same plasmid. We show that it can be used efficiently in C. glabrata, for both described outcomes of CRISPR‐Cas9‐induced chromosome breaks; nonhomologous end joining in the absence of a homologous repair template; and homologous recombination in the presence of such a template. This system therefore allows easy editing of the genome of C. glabrata, and its inducibility may allow identification of essential genes in this asexual yeast, where spore lethality cannot be observed, as well as the study of double‐strand break repair.

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CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles

Cited by 173 publications

References 51 publications

Decoding non-random mutational signatures at Cas9 targeted sites

Decoding non-random mutational signatures at Cas9 targeted sites

Cas9-induced large deletions and small indels are controlled in a convergent fashion

A new inducible CRISPR‐Cas9 system useful for genome editing and study of double‐strand break repair in Candida glabrata

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