A Rad51-Independent Pathway Promotes Single-Strand Template Repair in Gene Editing

Abstract

1 2 The Rad51/RecA family of recombinases perform a critical function in typical repair of double-3 strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded 4 DNA (dsDNA) template sequence to facilitate repair. However, repair of a DSB using single 5 stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene 6 editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-7 independent events in Saccharomyces cerevisiae in two different assays. Gene editing events 8 were carried out either by creating a DSB with the site-specific HO endonuclease and repairing 9 the DSB with 80-nt single-stranded oligonucleotides (ssODNs) or by using a bacterial retron 10 system that produces ssDNA templates in vivo in combination with DSBs created by Cas9. We 11show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the 12 Mre11-Rad50-Xrs2 (MRX) complex, but not Rad51, Rad54 or Rad55. Srs2 acts to prevent 13 overloading of Rad51 on the ssDNA filament, whereas Rad59 appears to alleviate the inhibition 14 of Rad51 on Rad52's strand annealing activity; thus, deletion of RAD51 suppresses both the 15 srs2Δ and rad59Δ phenotypes. This same suppression by rad51Δ of rad59Δ is found in another 16 DSB repair pathway, single strand annealing (SSA). In contrast, gene targeting using an 80-bp 17 dsDNA template of the same sequence is Rad51-dependent. We also examined SSTR events in 18 which the ssODN carried several mismatches. In the absence of the mismatch repair protein, 19Msh2, we found that the fate of mismatches carried on the ssDNA template are very different 20 at the 3' end, which can anneal directly to the resected DSB end, compared to the 5' end. We 21 also find that DNA polymerase Pold's 3' to 5' proofreading activity frequently excises a 22 Rdh54/Tid1 assume important roles. Rad51-independent BIR also requires the Mre11-Rad50-51Xrs2 complex, whereas Rad51-mediated events and SSA are merely delayed by the absence of 52 these proteins [18]. A third Rad51-independent pathway, another form of BIR, operates to 53 maintain telomeres in the absence of telomerase (known as Type II events). Here too, Rad59 54 and the MRX complex, as well as Rad52, are necessary, whereas the Type I Rad51-dependent 55 telomere maintenance pathway does not require either Rad59 or MRX [22,23,24]. Similarly, 56 DSB repair by intramolecular gene conversion involving short (33-bp) regions of homology is 57 inhibited by Rad51 and is dependent on the MRX complex, Rad59, and Rdh54 [25]. Rad51-58 independent BIR pathways are also dependent on the Srs2 helicase that antagonizes loading of 59 Rad51 onto resected DSB ends [21,26]. 60Use of the RNA-guided CRISPR/Cas9 endonuclease has revolutionized gene editing in 61 eukaryotic systems ranging from yeast to mammals [27,28,29]. Guided endonucleases are 62 programmed to create site-specific DSBs that can be repaired by providing a homologous 63 template [30,31]. One approach that has been shown to be an e...

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 3). As described in other eukaryotes (Gallagher and Haber, 2017;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 Fig.…”

“…Sequence analyses of the recovered Chlamydomonas colonies provided insights regarding the mechanism of DSB repair involving ssODN templates. As described in several eukaryotes (Gallagher and Haber, 2017;Kan et al, 2017;Paix et al, 2017;Boel et al, 2018;Harmsen et al, 2018;Richardson et al, 2018;Sansbury et al, 2019;Gallagher et al, 2020), the single-strand template repair (SSTR) model appears to explain best the repair of Cas9-induced DSBs with ssODNs as homologous donor DNA. In SSTR, ssODNs are used for the synthesis of complementary DNA, rather than being integrated into the genome, and the process is polarity sensitive and dependent, unlike other homologous repair mechanisms, on the Fanconi anemia pathway (Gallagher and Haber, 2017;Kan et al, 2017;Paix et al, 2017;Boel et al, 2018;Harmsen et al, 2018;Richardson et al, 2018).…”

“…In contrast, precise gene editing still occurs at relatively low frequencies or it is limited to specific (cell-wall-less) strains or experimental approaches (Ferenczi et al, 2017;Greiner et al, 2017;. In Chlamydomonas, as observed in land plants and other eukaryotes (Gallagher and Haber, 2017;Kan et al, 2017;Paix et al, 2017;Boel et al, 2018;Richardson et al, 2018;Sansbury et al, 2019;Capdeville et al, 2020;Gallagher et al, 2020), the repair of DSBs induced by CRISPR/Cas9 RNPs likely occurs by several distinct pathways, partly determined by the repair machinery expressed in each cell, the complexity of the DSB, and the nature of the DNA molecules involved in the repair. As described for some mammalian cell lines (Shy et al, 2016;Mitzelfelt et al, 2017), only a small subset of the population, in asynchronously grown Chlamydomonas, may be capable of HDR, possibly associated with being in a certain phase of the cell cycle (Angstenberger et al, 2020).…”

“…SSNs cause DNA double-strand breaks (DSBs) at specific sites, which can be repaired by alternative cellular mechanisms resulting in mutations or precise sequence changes. DSB repair by error-prone non-homologous 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, 2017; Capdeville et al, 2020; Gallagher et al, 2020; Scully 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, 2017; Paix et al, 2017; Capdeville et al, 2020; Gallagher et al, 2020; Scully et al, 2020).…”

Mechanisms preventing Break-Induced Replication during repair of two-ended DNA double-strand breaks

“…Both purified enzymes show annealing activity but only Rad52 can anneal complementary ssDNA in presence of RPA (Wu, Sugiyama et al, 2006). Rad59 interacts with Rad52 and stimulates annealing activity of Rad52 in suboptimal conditions, likely by mitigating the negative impact of Rad51 binding to Rad52 on annealing (Davis & Symington, 2001, Gallagher, Pham et al, 2020, Wu, Kantake et al, 2008. In cells, Rad59 seems to be particularly important for annealing of shorter and not completely homologous sequences (Sugawara, Ira et al, 2000).…”