Abstract
CRISPR/Cas9 screening has proven to be a versatile tool for genomics research. Based on unexpected results from a genome-wide screen, we developed a CRISPR/Cas9-mediated approach to mutagenesis, exploiting the allelic diversity generated by error-prone non-homologous end-joining (NHEJ) to identify novel gain-of-function and drug resistant alleles of the MAPK signaling pathway genes MEK1 and BRAF. We define the parameters of a scalable technique to easily generate cell populations containing thousands of endogenous allelic variants to map gene functions. Further, these results highlight an unexpected but important phenomenon, that Cas9-induced gain-of-function alleles are an inherent by-product of normal Cas9 loss-of-function screens and should be investigated during analysis of data from large-scale positive selection screens.
Highlights
Deciphering the functional consequences of DNA variation is a defining challenge of the genomic era, and CRISPR/Cas9 technology is the most promising and broadly-developed tool for facile genome engineering [1,2]
We introduced the sgRNA targeting MAP2K1 at the nucleotides encoding K59 or a control sgRNA targeting EGFP into A375 cells, treated with either vemurafenib or no selection for two weeks, harvested genomic DNA, and performed PCR with primers flanking the MAP2K1 target site followed by next-generation sequencing (Fig 1A)
Variant [9] and the Q56P variant, whereas overexpression of wild-type MEK1 did not produce appreciable resistance (Fig 2). These results show that CRISPR/Cas9-mediated mutagenesis of endogenous alleles coupled with strong positive selection can be used to discover novel gain-offunction protein variants and provide insight into regulatory domains
Summary
Deciphering the functional consequences of DNA variation is a defining challenge of the genomic era, and CRISPR/Cas9 technology is the most promising and broadly-developed tool for facile genome engineering [1,2]. These positive selection screens utilized vemurafenib, a BRAF inhibitor, and selumetinib, a MEK inhibitor, to identify sgRNAs that induce drug-resistance in cells and enrich in the cell population over time.
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