Exploiting the CRISPR/Cas9 PAM constraint for single-nucleotide resolution interventions

Yi Li; Saurabh Mendiratta; Kristina Ehrhardt; Neha Kashyap; Michael A. White; Leonidas Bleris

doi:10.1371/journal.pone.0144970

Exploiting the CRISPR/Cas9 PAM constraint for single-nucleotide resolution interventions

Yi Li, Saurabh Mendiratta, Kristina Ehrhardt, Neha Kashyap, Michael A. White, Leonidas Bleris

Research output: Contribution to journal › Article › peer-review

17 Scopus citations

Abstract

CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. An acute DNA binding constraint of the Cas9 protein is the Protospacer Adjacent Motif (PAM). Here we demonstrate that the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wildtype alleles. Specifically, we target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9 to a desired protospacer adjacent motif.

Original language	English (US)
Article number	e0144970
Journal	PloS one
Volume	11
Issue number	1
DOIs	https://doi.org/10.1371/journal.pone.0144970
State	Published - Jan 1 2016

ASJC Scopus subject areas

Biochemistry, Genetics and Molecular Biology(all)
Agricultural and Biological Sciences(all)
General

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abstract = "CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. An acute DNA binding constraint of the Cas9 protein is the Protospacer Adjacent Motif (PAM). Here we demonstrate that the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wildtype alleles. Specifically, we target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9 to a desired protospacer adjacent motif.",

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note = "Funding Information: This work was funded by the US National Institutes of Health (NIH) grants GM096271, CA170018, the US National Science Foundation (NSF) CAREER grant 1351354, the University of Texas at Dallas, and Robert Welch Foundation (I-1414). We would like to thank S. Lawson, M. Hasoon, T. Guinn and S. Lin for technical support. Publisher Copyright: {\textcopyright} 2016 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.",

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AU - Kashyap, Neha

AU - White, Michael A.

AU - Bleris, Leonidas

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Exploiting the CRISPR/Cas9 PAM constraint for single-nucleotide resolution interventions

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