Format

Send to

Choose Destination
PLoS One. 2017 Feb 23;12(2):e0172177. doi: 10.1371/journal.pone.0172177. eCollection 2017.

Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis.

Author information

1
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.
2
Department of Cancer Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
3
Molecular and Cellular Biology Program, Stony Brook University, Stony Brook, New York, United States of America.
4
W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Howard Hughes Medical Institute, Cold Spring Harbor, New York, United States of America.

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.

PMID:
28231254
PMCID:
PMC5322889
DOI:
10.1371/journal.pone.0172177
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center