title: Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing.
abstract: Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle, two orthogonal strategies for reducing off-target cleavage, truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs), could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally, we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR-Cas nucleases and therefore provide a highly specific platform for performing genome editing.
authors.affiliation: 1 Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital , Charlestown, Massachusetts.|2 Center for Computational and Integrative Biology, Massachusetts General Hospital , Charlestown, Massachusetts.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Wyvekens
authors.affiliation: 1 Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital , Charlestown, Massachusetts.|2 Center for Computational and Integrative Biology, Massachusetts General Hospital , Charlestown, Massachusetts.
authors.forename: Ved V
authors.initials: VV
authors.lastname: Topkar
authors.affiliation: 1 Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital , Charlestown, Massachusetts.|2 Center for Computational and Integrative Biology, Massachusetts General Hospital , Charlestown, Massachusetts.
authors.forename: Cyd
authors.initials: C
authors.lastname: Khayter
authors.affiliation: 1 Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital , Charlestown, Massachusetts.|2 Center for Computational and Integrative Biology, Massachusetts General Hospital , Charlestown, Massachusetts.|3 Department of Pathology, Harvard Medical School, Boston, Massachusetts.
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.affiliation: 1 Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital , Charlestown, Massachusetts.|2 Center for Computational and Integrative Biology, Massachusetts General Hospital , Charlestown, Massachusetts.|3 Department of Pathology, Harvard Medical School, Boston, Massachusetts.
authors.forename: Shengdar Q
authors.initials: SQ
authors.lastname: Tsai
mesh_terms: D001426:Bacterial Proteins / Q000502:physiology*; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D053595:Embryonic Stem Cells / Q000502:physiology; D004706:Endodeoxyribonucleases / Q000502:physiology*; D004720:Endonucleases / Q000502:physiology*; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000502:physiology*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; C058668:DNA modification methylase FokI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004706:Endodeoxyribonucleases; D004720:Endonucleases