Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity

Ian P. Winters, Shin-Heng Chiou, Nicole K. Paulk, Christopher D. McFarland, Pranav V. Lalgudi, Rosanna K. Ma, Leszek Lisowski, Andrew J. Connolly, Dmitri A. Petrov, Mark A. Kay and Monte M. Winslow ()
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Ian P. Winters: Stanford University School of Medicine
Shin-Heng Chiou: Stanford University School of Medicine
Nicole K. Paulk: Stanford University School of Medicine
Christopher D. McFarland: Stanford University
Pranav V. Lalgudi: Stanford University School of Medicine
Rosanna K. Ma: Stanford University School of Medicine
Leszek Lisowski: Stanford University School of Medicine
Andrew J. Connolly: Stanford University School of Medicine
Dmitri A. Petrov: Stanford University
Mark A. Kay: Stanford University School of Medicine
Monte M. Winslow: Stanford University School of Medicine

Nature Communications, 2017, vol. 8, issue 1, 1-16

Abstract: 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 Kras HDR 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.

Date: 2017
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DOI: 10.1038/s41467-017-01519-y

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