title: Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.
abstract: Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.
authors.affiliation: ISIS Pharmaceuticals, Carlsbad, CA 92010;
authors.forename: Meghdad
authors.initials: M
authors.lastname: Rahdar
authors.affiliation: Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093.
authors.forename: Moira A
authors.initials: MA
authors.lastname: McMahon
authors.affiliation: ISIS Pharmaceuticals, Carlsbad, CA 92010;
authors.forename: Thazha P
authors.initials: TP
authors.lastname: Prakash
authors.affiliation: ISIS Pharmaceuticals, Carlsbad, CA 92010;
authors.forename: Eric E
authors.initials: EE
authors.lastname: Swayze
authors.affiliation: ISIS Pharmaceuticals, Carlsbad, CA 92010; fbennett@isisph.com dcleveland@ucsd.edu.
authors.forename: C Frank
authors.initials: CF
authors.lastname: Bennett
authors.affiliation: Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093 fbennett@isisph.com dcleveland@ucsd.edu.
authors.forename: Don W
authors.initials: DW
authors.lastname: Cleveland
mesh_terms: D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005813:Genes, Synthetic; D005818:Genetic Engineering; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D012313:RNA
keywords: CRISPR/Cas9; genome editing; scrRNA; synthetic crRNA