A Rad51-independent pathway promotes single-strand template repair in gene editing
Fig 5
SSTR initiated by retron-Cas9 utilizes a Rad51-indepndent repair pathway.
A) The retron system utilizes a modified Cas9 gRNA that tethers the ssDNA donor template to the RNA scaffolding of the Cas9 protein. Successful SSTR at the MAT locus results in the insertion of an XhoI restriction site, as in Fig 1. SSTR at the lys5 locus repairs a 5-bp insertion in the lys5 locus, resulting in Lys+ recombinants. B) Efficiency of the Retron-Cas9 system at two chromosomal locations. Cells were plated onto URA- plates with dextrose (non-induction) and URA- with galactose (induction) media. At MAT, the percent gene editing was determined by PCR and XhoI digest of induction survivors as described in Methods. At lys5, the percentage of gene editing was determined by replica plating URA-Gal survivors onto Lys- media. The resulting plate count over plate counts of URA- non-induction media results in % gene editing. C) Effect of recombination mutants on retron-Cas9 SSTR gene editing. After induction of the retron system on galactose-containing media, survivors were replica plates to Lys- media. The frequency of Lys+ colonies was calculated as a percentage of total cells plated and normalized to wild type. Significance was determined using two-tailed t-tests compared to WT, using the two-stage Benjamini, Krieger, and Yekutieli false discovery rate approach [89], * p ≤ 0.01, ** p ≤ 0.001, *** p ≤ 0.0001. Error bars refer to the standard error of the mean. n = 3. rad59Δ compared to rad59Δ rad51Δ p = 0.009.