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      Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells

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          Abstract

          The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases 1 are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9) 2– 5 . We also report that four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Martin Jinek, Krzysztof Chylinski, Ines Fonfara … (2012)
          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
          Article 0 comments Cited 2965 times – based on 0 reviews     Review now
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            Multiplex genome engineering using CRISPR/Cas systems.

            Le Cong, F. Ran, David T. Cox … (2013)
            Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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              RNA-guided human genome engineering via Cas9.

              P. Mali, L. YANG, K. Esvelt … (2013)
              Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
              Article 0 comments Cited 1414 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-journal-id): 9604648
                Journal ID (pubmed-jr-id): 20305
                Journal ID (nlm-ta): Nat Biotechnol
                Journal ID (iso-abbrev): Nat. Biotechnol.
                Title: Nature biotechnology
                ISSN (Print): 1087-0156
                ISSN (Electronic): 1546-1696
                Publication date Nihms-submitted: 25 May 2016
                Publication date (Electronic): 27 June 2016
                Publication date (Print): August 2016
                Publication date PMC-release: 27 December 2016
                Volume: 34
                Issue: 8
                Pages: 869-874
                Affiliations
                [1 ]Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA
                [2 ]Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA
                [3 ]Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA
                [4 ]Department of Pathology, Harvard Medical School, Boston, MA, USA
                [5 ]Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA
                Author notes
                [6]

                These authors contributed equally to this work

                Article
                Manuscript ID: NIHMS789773
                DOI: 10.1038/nbt.3620
                PMC ID: 4980201
                PubMed ID: 27347757
                SO-VID: 0cb94c94-dbb5-4498-b598-f99788cf9d25
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                Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

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