Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions

PloS One
Yi LiLeonidas Bleris

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 wild-type 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.

References

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Jan 5, 2013·Science·Prashant MaliGeorge M Church
Apr 23, 2014·Nature Biotechnology·Xuebing WuPhillip A Sharp
May 6, 2014·Nucleic Acids Research·Tom Schreiber, Ulla Bonas
Jun 17, 2014·ACS Synthetic Biology·Richard MooreLeonidas Bleris
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Citations

Oct 14, 2016·Nucleic Acids Research·Chance M NowakLeonidas Bleris
Jan 28, 2017·Molecular Therapy : the Journal of the American Society of Gene Therapy·Alex Mas MonteysBeverly L Davidson
Nov 25, 2017·Scientific Reports·Kathleen A ChristieC B Tara Moore
Oct 20, 2018·Journal of Cellular and Molecular Medicine·Li ZhouJinming Li
May 2, 2019·BioTechniques·Claude Van CampenhoutVéronique Kruys
Nov 2, 2019·Genes·Enrique Hernández-LemusSoledad Ochoa
Jun 25, 2016·Bioengineered·Jian-Hua ZhangWilliam F Simonds
Dec 28, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Taegeun BaeJunho K Hur
Oct 28, 2019·Biophysical Journal·Yi LiFaruck Morcos
Nov 29, 2020·Molecular Therapy : the Journal of the American Society of Gene Therapy·Roy Rabinowitz, Daniel Offen

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Methods Mentioned

BETA
Flow
fluorescence microcopy
transfection
fluorescence microscopy
PCR
transfections
Assay

Software Mentioned

Slidebook
Protospacer
FinchTV
FlowJo
Chroma

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