Figure S17. Generation of a point mutation in Rims1 with a lssDNA donors. (a) PCR amplification of region of interest with Rims1-F2 and Rims1-R2 primers (647 bp) from biopsies taken from the F0 animals. Animal IDs are shown. + is positive control amplified from an unrelated WT animal. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). (b) Sequencing of amplicon obtained from the Rims1-lss-2, Rims1-lss-20, Rims1-lss-21 and Rims1-lss-36 animals: point mutation is observed (blue highlight) when sequencing the Rims1-F2 primer. (c) The table details the F0 animals obtained for generation of Rims1 mutant with lssDNA donors. The ID, outcome of sequencing the region of interest and the conclusion for each individual are shown. (d) The table details the first litter obtained by mating Rims1-lss-36 with a WT mouse. The ID, outcome of sequencing the region of interest, copy counting of the region of interest and conclusion for each individual are shown. (e) PCR amplification of region of interest with Rims1-F3 and Rims1-R3 primers (647 bp) from biopsies taken from Rims-lss-36’s offspring. Animal IDs are shown. + is a positive control amplified from an unrelated WT animal. L2 = 100 bp DNA molecular weight ladder (thick bands are 1000 and 500 bp). (f) Sequencing of amplicon obtained from Rims1-lss-36.1a, legitimate repair observed (blue highlight) when sequencing both directions (Rims1-F3 and Rims1-R3 primers). (g) Alignment of Rims1-lss-36-1a offspring, legitimate repair aligned against WT allele. R655H coding change highlighted in red. Grey background with red text highlights silent mutations introduced by long donor. (PNG 1287 kb
