title: Creation of Novel Protein Variants with CRISPR/Cas9-Mediated Mutagenesis: Turning a Screening By-Product into a Discovery Tool.
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.
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA, United States of America.
authors.forename: Katherine F
authors.initials: KF
authors.lastname: Donovan
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA, United States of America.
authors.forename: Mudra
authors.initials: M
authors.lastname: Hegde
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA, United States of America.
authors.forename: Meagan
authors.initials: M
authors.lastname: Sullender
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA, United States of America.
authors.forename: Emma W
authors.initials: EW
authors.lastname: Vaimberg
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA, United States of America.
authors.forename: Cory M
authors.initials: CM
authors.lastname: Johannessen
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA, United States of America.
authors.forename: David E
authors.initials: DE
authors.lastname: Root
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA, United States of America.
authors.forename: John G
authors.initials: JG
authors.lastname: Doench
mesh_terms: D000483:Alleles; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D059766:DNA End-Joining Repair; D015723:Gene Library; D057809:HEK293 Cells; D006801:Humans; D054643:INDEL Mutation; D007211:Indoles / Q000494:pharmacology; D048369:MAP Kinase Kinase 1 / Q000737:chemistry / Q000235:genetics*; D016296:Mutagenesis; D010641:Phenotype; D015202:Protein Engineering / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016415:Sequence Alignment; D013449:Sulfonamides / Q000494:pharmacology; D014161:Transduction, Genetic; D000077484:Vemurafenib
chemical_list: D007211:Indoles; D000094704:RNA, Guide, CRISPR-Cas Systems; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; D000077484:Vemurafenib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human