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

Ipsaro, J. J., Shen, C., Arai, E., Xu, Y., Kinney, J. B., Joshua-Tor, L., Vakoc, C. R., Shi, J. (2017) Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis. PLoS One, 12 (2). e0172177. ISSN 1932-6203

URL: https://www.ncbi.nlm.nih.gov/pubmed/28231254
DOI: 10.1371/journal.pone.0172177

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.

Item Type: Paper
Subjects: diseases & disorders > cancer
Investigative techniques and equipment > CRISPR-Cas9
diseases & disorders > cancer > drugs and therapies
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > mutations > mutagenesis
CSHL Authors:
Communities: CSHL labs > Joshua-Tor lab
CSHL labs > Kinney lab
CSHL labs > Vakoc lab
Depositing User: Matt Covey
Date Deposited: 02 Mar 2017 22:05
Last Modified: 02 Mar 2017 22:05
Related URLs:
URI: http://repository.cshl.edu/id/eprint/34139

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