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Abstract
Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded KrasHDR alleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.
Highlights
Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth
To investigate the oncogenic function of diverse point mutations in vivo in a quantitative and systematic manner, we developed a platform for somatic AAV/Cas9-mediated homology-directed repair (HDR) that incorporates DNA barcoding and high-throughput sequencing in autochthonous mouse models of several cancer types (Fig. 1)
The KrasHDR template contained the genomic sequence flanking the second exon of Kras and either wild-type (WT) Kras exon 2 or exon 2 with one of 12 single-nucleotide nonsynonymous mutations in codon 12 or 13 (Fig. 2a)
Summary
Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. We present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and highthroughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. Tumors in genetically engineered mice are driven by defined mutations expressed at physiological levels and develop within their natural context Tumors initiated in these autochthonous mouse models recapitulate the gene expression programs and histopathological progression of human cancers, including the development of invasive and metastatic disease[4]. Cas9-mediated HDR has enabled precise genomic editing in cell lines and organoids, and HDR has been used in mice to introduce reporter genes, correct disease alleles, and introduce targeted point mutations in several adult tissues[4,12,13,14,15,16,17]
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