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High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

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      Abstract

      CRISPR RNA-guided endonucleases (RGENs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of Cas9-based RGENs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We readily detected off-target alterations induced by four out of six RGENs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbor up to five mismatches and many are mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGENs are highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.

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      Most cited references 20

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      Multiplex genome engineering using CRISPR/Cas systems.

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

        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.
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          RNA-guided human genome engineering via Cas9.

          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.
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            Author and article information

            Affiliations
            [1 ]Molecular Pathology Unit, Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA, Massachusetts General Hospital, Charlestown, MA, USA
            [2 ]Department of Pathology, Harvard Medical School, Boston, MA, USA
            [3 ]Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, USA
            Author notes
            Journal
            9604648
            20305
            Nat Biotechnol
            Nat. Biotechnol.
            Nature biotechnology
            1087-0156
            1546-1696
            12 July 2013
            23 June 2013
            September 2013
            01 March 2014
            : 31
            : 9
            : 822-826
            23792628
            3773023
            10.1038/nbt.2623
            NIHMS488397

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            Funding
            Funded by: National Institute of General Medical Sciences : NIGMS
            Award ID: R01 GM088040 || GM
            Funded by: National Human Genome Research Institute : NHGRI
            Award ID: P50 HG005550 || HG
            Funded by: National Institute of General Medical Sciences : NIGMS
            Award ID: DP1 GM105378 || GM
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            Biotechnology

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