A Rad51-independent pathway promotes single-strand template repair in gene editing

Abstract

The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo . We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52’s strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3’ side of the ssODN which can anneal directly to the resected DSB end compared to the 5’ end. In addition DNA polymerase Polδ’s 3’ to 5’ proofreading activity frequently excises a mismatch very close to the 3’ end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.

Mapping the Genetic Landscape of DNA Double-strand Break Repair

“…Strikingly, across each of these donors, genes encoding components of the MutSα/MutLα DNA mismatch repair (MMR) complexes ( MSH2 , MSH6 , MLH1 , and PMS2 ) strongly promoted some scarless SSTR outcomes while suppressing others, with the suppressed set biased for outcomes missing SNVs from the 3’ ends of ssODNs (Figure S7C-S7E). These results are consistent with observations from previous studies, which proposed that MMR activity installs SNVs encoded on the 3’ ends of ssODNs by “fixing” mismatches established upon donor annealing 78, 81, 82 .…”

Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell

“…Previous studies using I-SceI breaks have shown that single-strand templated repair (SSTR) is Rad51-independent in yeast ( 32 ). A recent publication also supports a Rad51-independent mechanism for SSTR after Cas9 breaks, and has found that SSTR is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex in yeast ( 33 ). Similarly, a CRISPR screen for SSTR suggests no involvement of RAD51 and BRCA2 in ssODN integration ( 34 ).…”

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

“…However, we surmise that the remaining examined colonies were also edited by homology-directed mechanisms, since we have not recovered any spontaneous herbicide-resistant mutant in any of our negative control experiments ( Supplemental Table S3 ). As described in other eukaryotes ( Gallagher and Haber, 2018 ; Paix et al, 2017 ; Boel et al, 2018 ; Gallagher et al, 2020 ), we expected that Cas9-induced DSBs in Chlamydomonas would be repaired by the single-strand template repair (SSTR) mechanism (see “Discussion”). Following this model ( Supplemental Figure S4 ), the PPX1 ssODN would be used as a homologous template for DNA synthesis primed by the 3′ ending strand on the left side of the cleavage site.…”

“…DSB repair by error-prone nonhomologous end-joining (NHEJ), including canonical and alternative pathways, may result in insertions/deletions (i.e. indels) and/or missense/nonsense mutations at the target sites ( Rodgers and McVey, 2016 ; Gallagher and Haber, 2018 ; Scully et al, 2019 ; Capdeville et al, 2020 ; Gallagher et al, 2020 ). Alternatively, homology directed repair (HDR) in the presence of template donor DNA, which can also occur by several pathways, may result in precise sequence changes ( Rodgers and McVey, 2016 ; Gallagher and Haber, 2018 ; Paix et al, 2017 ; Scully et al, 2019 ; Capdeville et al, 2020 ; Gallagher et al, 2020 ).…”