title: Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis.
abstract: Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development.
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Ipsaro
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.
authors.forename: Chen
authors.initials: C
authors.lastname: Shen
authors.affiliation: Department of Cancer Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Eri
authors.initials: E
authors.lastname: Arai
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.|Molecular and Cellular Biology Program, Stony Brook University, Stony Brook, New York, United States of America.
authors.forename: Yali
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.
authors.forename: Justin B
authors.initials: JB
authors.lastname: Kinney
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.|W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Howard Hughes Medical Institute, Cold Spring Harbor, New York, United States of America.
authors.forename: Leemor
authors.initials: L
authors.lastname: Joshua-Tor
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Vakoc
authors.affiliation: Department of Cancer Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Junwei
authors.identifier: http://orcid.org/0000-0002-8427-6316
authors.initials: J
authors.lastname: Shi
mesh_terms: D000483:Alleles; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001562:Benzimidazoles / Q000494:pharmacology; D064113:CRISPR-Cas Systems; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D019008:Drug Resistance, Neoplasm; D000071221:Enhancer of Zeste Homolog 2 Protein / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D054643:INDEL Mutation; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D016296:Mutagenesis; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D001562:Benzimidazoles; C583893:EPZ-5676; C473477:DOT1L protein, human; D008780:Methyltransferases; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D011495:Histone-Lysine N-Methyltransferase