Fig. 3

AAV/Cas9-mediated somatic HDR initiates oncogenic Kras-driven lung tumors. a Schematic of the experiment to introduce point mutations and a DNA barcode into the endogenous Kras locus of lung epithelial cells in Rosa26 ^LSL-Tomato ;H11 ^LSL-Cas9 (T;H11 ^LSL-Cas9), p53 ^flox/flox ;T;H11 ^LSL-Cas9 (PT;H11 ^LSL-Cas9), and Lkb1 ^flox/flox ;T;H11 ^LSL-Cas9 (LT;H11 ^LSL-Cas9) mice by intratracheal administration of AAV-Kras ^HDR/sgKras/Cre (8.4 × 10¹⁰ vector genomes per mouse). b Representative fluorescence and histology images of Tomato^positive lung tumors in LT;H11 ^LSL-Cas9, PT;H11 ^LSL-Cas9, and T;H11 ^LSL-Cas9 mice transduced with AAV-Kras ^HDR/sgKras/Cre. Scale bars = 5 mm. c Quantification of lung tumors in the indicated genotypes of mice transduced with the indicated AAV vectors (with and without sgKras). Each dot represents one mouse. d Representative FACS plot showing Tomato^positive DTCs in the pleural cavity of an LT;H11 ^LSL-Cas9 mouse with AAV-Kras ^HDR/sgKras/Cre-initiated lung tumors. Plot shows forward scatter/side scatter (SSC)-gated viable cancer cells (DAPI/CD45/CD31/F4-80/Ter119^negative). e Histology of lymphatic metastases from AAV-Kras ^HDR/sgKras/Cre-initiated lung tumors in PT;H11 ^LSL-Cas9 mice. Scale bar = 50 µm. f Number of AAV-Kras ^HDR/sgKras/Cre-transduced mice of each genotype that had disseminated tumors cells in their pleural cavity (DTCs > 10) or metastases out of the total number of mice analyzed. g Kras ^LSL-G12D/+ ;LT (KLT) and Kras ^LSL-G12D/+ ;PT (KPT) mice transduced with a 1:10,000 dilution of AAV-Kras ^HDR/sgKras/Cre developed approximately half as many tumors as the PT;H11 ^LSL-Cas9 and LT;H11 ^LSL-Cas9 mice transduced with undiluted virus. If all Kras ^HDR alleles in the AAV-Kras ^HDR/sgKras/Cre library are oncogenic, this suggests that AAV/Cas9-mediated HDR occurs in ~0.02% of transduced cells. Alternatively, if only 20% of the mutant alleles in the AAV-Kras ^HDR/sgKras/Cre library are oncogenic, this suggests that HDR occurs in ~0.1% of transduced cells. h Diverse HDR-generated oncogenic Kras alleles in individual lung tumors dissected from LT;H11 ^LSL-Cas9 and PT;H11 ^LSL-Cas9 mice transduced with AAV-Kras ^HDR/sgKras/Cre. Number of tumors with each allele is indicated. Alleles that were not identified in any lung tumors are not shown. For the Kras ^HDR alleles identified, Bonferroni corrected p values for likelihood of enrichment relative to WT are shown (Fisher’s exact test generalized for structural zeros; see Methods section)