Ipsaro, J. J., Shen, C., Arai, E., Xu, Y., Kinney, J. B., Joshua-Tor, L., Vakoc, C. R., Shi, J. (February 2017) Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis. PLoS One, 12 (2). e0172177. ISSN 1932-6203
Preview |
PDF (Paper)
Kinney et al 2017.pdf - Published Version Download (1MB) | Preview |
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 Cancer Center Program > Gene Regulation and Cell Proliferation CSHL labs > Joshua-Tor lab CSHL labs > Kinney lab CSHL labs > Vakoc lab CSHL Cancer Center Program > Cancer Genetics and Genomics Program CSHL Cancer Center Program > Gene Regulation and Inheritance Program |
| Depositing User: | Matt Covey |
| Date: | 23 February 2017 |
| Date Deposited: | 02 Mar 2017 22:05 |
| Last Modified: | 06 Jul 2021 19:32 |
| PMCID: | PMC5322889 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/34139 |
Actions (login required)
 |
Administrator's edit/view item |