Fig. 1
A cancer modeling platform that integrates AAV/Cas9-mediated somatic HDR with tumor barcoding and sequencing to enable the rapid introduction and functional investigation of putative oncogenic point mutations in vivo. a–d Schematic of the pipeline to quantitatively measure the in vivo oncogenicity of a panel of defined point mutations. a A library of AAV vectors is generated such that each AAV contains: (1) a template for homology-directed repair (HDR) containing a putatively oncogenic point mutation and a random DNA barcode encoded in the adjacent wobble bases; (2) an sgRNA targeting the desired endogenous locus to enhance HDR; and (3) Cre-recombinase to activate a conditional Cas9 allele (H11 LSL-Cas9) and other Cre-dependent alleles in genetically engineered mice. b The AAV library is delivered to a tissue of interest. c Following transduction, a subset of cells undergo AAV/Cas9-mediated HDR in which the locus of interest is cleaved by Cas9 at the sgRNA target site and repaired using the AAV HDR template. This results in the introduction of the desired point mutation and a unique DNA barcode at the targeted locus. d Somatic cells engineered with a point mutation may develop into de novo tumors if the introduced mutation is sufficient to initiate tumorigenesis and drive tumor growth. Two independent approaches can be used to analyze tumors: (1) the targeted region in individual tumors can be sequenced to characterize both alleles of the targeted gene, or (2) next-generation sequencing of the targeted region can be used to determine the number, size, and genotype of each tumor directly from bulk tissue in a quantitative and multiplexed manner
