Fig. 2

Design and validation of an AAV targeting vector library to introduce all Kras codon 12 and 13 single-nucleotide non-synonymous point mutations into somatic mouse cells in a multiplexed manner. a AAV vector pool for Cas9-mediated HDR into the endogenous Kras locus (AAV-Kras ^HDR/sgKras/Cre). Each vector contained an HDR template with 1 of 12 non-synonymous Kras mutations at codons 12 and 13 (or wild-type Kras), silent mutations within the PAM (boxed sequence) and sgRNA homology region (PAM*), and a random 8-nucleotide barcode within the wobble positions of the adjacent codons for stable DNA barcoding of individual tumors. b Representation of each Kras ^HDR allele in the AAV-Kras ^HDR/sgKras/Cre plasmid library. c Diversity of the barcode region in the AAV-Kras ^HDR/sgKras/Cre plasmid library. d Schematic of the experiment to test for HDR bias. A Cas9-expressing cell line was transduced with AAV-Kras ^HDR/sgKras/Cre and Kras ^HDR alleles were sequenced to quantify HDR events. e Schematic of the PCR strategy to specifically amplify Kras ^HDR alleles introduced into the genome via HDR. Forward primer 1 (F1) binds to the sequence containing the 3 PAM* mutations, while reverse primer 1 (R1) binds the endogenous Kras locus, outside the sequence present in the homology arm of the Kras ^HDR template. F2 binds to the Illumina adaptor added by F1, R2 binds to a region near exon 2, and R3 binds to the Illumina adapter added in the same reaction by R2. f Frequency of HDR events for each Kras ^HDR allele plotted against the initial frequency of each Kras mutant allele in the AAV-Kras ^HDR/sgKras/Cre plasmid library. High correlation between the initial plasmid library and the representation of mutant Kras alleles following HDR suggests little to no HDR bias