@article {Gallagher2020.02.24.962423, author = {Danielle N. Gallagher and Nhung Pham and Annie M. Tsai and Abigail N. Janto and Jihyun Choi and Grzegorz Ira and James E. Haber}, title = {A Rad51-Independent Pathway Promotes Single-Strand Template Repair in Gene Editing}, elocation-id = {2020.02.24.962423}, year = {2020}, doi = {10.1101/2020.02.24.962423}, publisher = {Cold Spring Harbor Laboratory}, 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 facilitate 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 in two different assays. Gene editing events were carried out either by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs) or by using a bacterial retron system that produces ssDNA templates in vivo in combination with DSBs created by Cas9. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but not Rad51, Rad54 or Rad55. Srs2 acts to prevent overloading of Rad51 on the ssDNA filament, whereas Rad59 appears to alleviate the inhibition of Rad51 on Rad52{\textquoteright}s strand annealing activity; thus, deletion of RAD51 suppresses both the srs2Δ and rad59Δ phenotypes. This same suppression by rad51Δ of rad59Δ is found in another DSB repair pathway, single strand annealing (SSA). In contrast, gene targeting using an 80-bp dsDNA template of the same sequence is Rad51-dependent. We also examined SSTR events in which the ssODN carried several mismatches. In the absence of the mismatch repair protein, Msh2, we found that the fate of mismatches carried on the ssDNA template are very different at the 3{\textquoteright} end, which can anneal directly to the resected DSB end, compared to the 5{\textquoteright} end. We also find that DNA polymerase Polδ{\textquoteright}s 3{\textquoteright} to 5{\textquoteright} proofreading activity frequently excises a mismatch close to the 3{\textquoteright} end of the template, similar to its removal of heterologies close to the 3{\textquoteright} invading end of the DSB. We further report that SSTR is accompanied by a 600-fold increase in mutations in a region adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.}, URL = {https://www.biorxiv.org/content/early/2020/02/24/2020.02.24.962423}, eprint = {https://www.biorxiv.org/content/early/2020/02/24/2020.02.24.962423.full.pdf}, journal = {bioRxiv} }