Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

Paix, Alexandre; Folkmann, Andrew W.; Goldman, Daniel; Kulaga, Heather; Grzelak, Michael; Rasoloson, Dominique; Paidemarry, Supriya; Green, Rachel; Reed, Randall R.; Seydoux, Géraldine

doi:10.1101/161109

Cited by 46 publications

(85 citation statements)

References 30 publications

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“…We designed 10 sgRNAs leading to cleavage between -36 and +40 nt of a single SEC61B insertion site in HEK293T cells. Following the integration of a 2xGFP11 sequence (165 nt), we observed maximal efficiency using sgRNAs cutting within ±10 nt of the integration site ( Figure 3F), confirming other reports (20,21).…”

Section: Design Rules For Ssdna Hdr Donorssupporting

confidence: 90%

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Design and specificity of long ssDNA donors for CRISPR-based knock-in

Pessino

et al. 2017

Preprint

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CRISPR/Cas technologies have transformed our ability to manipulate genomes for research and genebased therapy. In particular, homology-directed repair after genomic cleavage allows for precise modification of genes using exogenous donor sequences as templates. While both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) forms of donors have been used as repair templates, a systematic comparison of the performance and specificity of repair using ssDNA versus dsDNA donors is still lacking. Here, we describe an optimized method for the synthesis of long ssDNA templates and demonstrate that ssDNA donors can drive efficient integration of gene-sized reporters in human cell lines. We next define a set of rules to maximize the efficiency of ssDNAmediated knock-in by optimizing donor design. Finally, by comparing ssDNA donors with equivalent dsDNA sequences (PCR products or plasmids), we demonstrate that ssDNA templates have a unique advantage in terms of repair specificity while dsDNA donors can lead to a high rate of off-target integration. Our results provide a framework for designing high-fidelity CRISPR-based knock-in experiments, in both research and therapeutic settings.

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Section: Design Rules For Ssdna Hdr Donorssupporting

confidence: 90%

“…Of note, we found variable ssDNA-mediated integration efficiencies in HEK293T and K562 cells (compare Figures 3A and 3C). The HDR pathway used by ssDNA templates is still unclear but likely differs from classical RAD51-dependent repair (21,(26)(27)(28), and might be active at different levels across cell lines.…”

Section: Discussionmentioning

confidence: 99%

Design and specificity of long ssDNA donors for CRISPR-based knock-in

Pessino

et al. 2017

Preprint

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“…Davis and Maizels 2016) and in the context of repair from Cas9 DSBs (Kan et al 2017;Paix et al 2017)) and meganucleases (Kan et al 2014).…”

Section: Precise Repair From Homologous Single-stranded Oligonucleotimentioning

confidence: 99%

Strategies for Efficient Genome Editing Using CRISPR-Cas9

Farboud

Severson

Meyer

2018

Preprint

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The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and non-model organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes, both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-stranded breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans. We devised strategies to insert long (10 kb) exogenous sequences or incorporate multiple nucleotide substitutions at considerable distance from DSBs. We expanded the repertoire of co-conversion markers appropriate for diverse nematode species. These selectable markers enable rapid identification of Cas9-edited animals also likely to carry edits in desired targets. Lastly, we explored the timing, location, frequency, sex-dependence, and categories of DSB repair events by developing loci with allele-specific Cas9 targets that can be contributed during mating from either male or hermaphrodite germ cells. Our studies revealed a striking difference in editing efficiency between maternally and paternally contributed genomes. Furthermore, imprecise repair and precise repair from exogenous repair templates occur with high frequency before and after fertilization. Our strategies enhance Cas9 targeting efficiency, lend insight into the timing and mechanisms of DSB repair, and establish guidelines for achieving predictable precise and imprecise repair outcomes with high frequency.

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“…CRISPR/Cas9-mediated homologous recombination (HR) was used to mimic the candidate mutation of MA lines 296, 450, 488 and 516. HR was performed using singlestranded DNA oligonucleotide repair templates (ssDORT) with 35 bp 5' and 3' homology arms, following a combination of previously described methods (Paix et al, 2017b, Paix et al, 2017a, Dokshin et al, 2018 The missense mutation in codon 59 from an asparagine (AAT) to a histidine (CAT) of sfrp-1 in C. briggsae (as found in MAL296) was edited using a crRNA guide (crRNA.sfrp-1.E2.g1) that induces a DSB 10 bp from the target region and a ssDORT (sfrp-1.E2.rt1) with the missense mutation and eight silent mutations. To control for the eight silent mutations, we generated control lines with another ssDORT (sfrp-1.E2.rt2) that only has the silent mutations.…”

Section: Crispr/cas9 Genome Editingmentioning

confidence: 99%

A broad mutational target explains an evolutionary trend

Besnard

Picao-Osorio

Dubois

et al. 2019

Preprint

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An evolutionary trend, the rapid evolution of a trait in a group of organisms, can in some cases be explained by the mutational variance, the propensity of a phenotype to change under spontaneous mutation. However, the causes of high mutational variance are still elusive. For some morphological traits, fast evolution was shown to depend on the high mutation rate of one or few underlying loci with short tandem repeats. Here, we investigate the case of the fastest evolving cell fate among vulva precursor cells in Caenorhabditis nematodes, that of the cell called 'P3.p'. For this, we combine mutation accumulation lines, whole-genome sequencing, genetic linkage analysis of the phenotype in recombinant lines, and candidate testing through mutant and CRISPR genome editing to identify causal mutations and the corresponding loci underlying the high mutational variance of P3.p. We identify and validate molecular lesions responsible for changes in this cell's phenotype during a mutation accumulation experiment. We find that these loci do not present any characteristics of a high mutation rate, are scattered across the genome and belong to distinct biological pathways. Our data instead indicate that a broad mutational target size is the cause of the high mutational variance and of the corresponding evolutionary trend.

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Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

Cited by 46 publications

References 30 publications

Design and specificity of long ssDNA donors for CRISPR-based knock-in

Design and specificity of long ssDNA donors for CRISPR-based knock-in

Strategies for Efficient Genome Editing Using CRISPR-Cas9

A broad mutational target explains an evolutionary trend

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