title: Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions.
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.
authors.affiliation: Bioengineering Department, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United States of America.|Center for Systems Biology, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United States of America.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390, United States of America.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Mendiratta
authors.affiliation: Bioengineering Department, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United States of America.|Center for Systems Biology, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United States of America.
authors.forename: Kristina
authors.initials: K
authors.lastname: Ehrhardt
authors.affiliation: Bioengineering Department, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United States of America.|Center for Systems Biology, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United States of America.
authors.forename: Neha
authors.initials: N
authors.lastname: Kashyap
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390, United States of America.
authors.forename: Michael A
authors.initials: MA
authors.lastname: White
authors.affiliation: Bioengineering Department, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United States of America.|Center for Systems Biology, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United States of America.|Electrical Engineering Department, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United States of America.
authors.forename: Leonidas
authors.initials: L
authors.lastname: Bleris
mesh_terms: D001562:Benzimidazoles / Q000494:pharmacology; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D057809:HEK293 Cells; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: C517975:AZD 6244; D001562:Benzimidazoles; C117307:KRAS protein, human; D000094704:RNA, Guide, CRISPR-Cas Systems; D012313:RNA; D016283:Proto-Oncogene Proteins p21(ras)