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.
MeSH terms
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Alleles
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CRISPR-Cas Systems*
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Cell Line, Tumor
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Clustered Regularly Interspaced Short Palindromic Repeats
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DNA End-Joining Repair
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Gene Library
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HEK293 Cells
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Humans
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INDEL Mutation
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Indoles / pharmacology
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MAP Kinase Kinase 1 / chemistry
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MAP Kinase Kinase 1 / genetics*
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Mutagenesis*
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Phenotype
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Protein Engineering / methods*
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Proto-Oncogene Proteins B-raf / chemistry
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Proto-Oncogene Proteins B-raf / genetics*
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RNA, Guide, CRISPR-Cas Systems / genetics
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Recombinant Fusion Proteins / chemistry
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Sequence Alignment
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Sulfonamides / pharmacology
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Transduction, Genetic
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Vemurafenib
Substances
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Indoles
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RNA, Guide, CRISPR-Cas Systems
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Recombinant Fusion Proteins
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Sulfonamides
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Vemurafenib
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BRAF protein, human
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Proto-Oncogene Proteins B-raf
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MAP Kinase Kinase 1
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MAP2K1 protein, human
Grants and funding
This study was funded by the Next Generation Fund at the Broad Institute of MIT and Harvard (JGD, Merkin Institute Fellow). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.