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      Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis

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          Significance

          Genome engineering in human pluripotent stem cells holds great promise for biomedical research and regenerative medicine, but it is very challenging. Recently, an RNA-guided nuclease system called clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) has been applied to genome engineering, greatly increasing the efficiency of genome editing. Here, using a CRISPR-Cas system identified in Neisseria meningitidis , which is distinct from the commonly used Streptococcus pyogenes system, we demonstrate efficient genome engineering in human pluripotent stem cells. Our study could have a tremendous impact in regenerative medicine.

          Abstract

          Genome engineering in human pluripotent stem cells (hPSCs) holds great promise for biomedical research and regenerative medicine. Recently, an RNA-guided, DNA-cleaving interference pathway from bacteria [the type II clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) pathway] has been adapted for use in eukaryotic cells, greatly facilitating genome editing. Only two CRISPR-Cas systems (from Streptococcus pyogenes and Streptococcus thermophilus ), each with their own distinct targeting requirements and limitations, have been developed for genome editing thus far. Furthermore, limited information exists about homology-directed repair (HDR)-mediated gene targeting using long donor DNA templates in hPSCs with these systems. Here, using a distinct CRISPR-Cas system from Neisseria meningitidis , we demonstrate efficient targeting of an endogenous gene in three hPSC lines using HDR. The Cas9 RNA-guided endonuclease from N. meningitidis (NmCas9) recognizes a 5′-NNNNGATT-3′ protospacer adjacent motif (PAM) different from those recognized by Cas9 proteins from S. pyogenes and S. thermophilus (SpCas9 and StCas9, respectively). Similar to SpCas9, NmCas9 is able to use a single-guide RNA (sgRNA) to direct its activity. Because of its distinct protospacer adjacent motif, the N. meningitidis CRISPR-Cas machinery increases the sequence contexts amenable to RNA-directed genome editing.

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          Most cited references43

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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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            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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              • Abstract: found
              • Article: not found

              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

                Journal
                Proceedings of the National Academy of Sciences
                Proc. Natl. Acad. Sci. U.S.A.
                Proceedings of the National Academy of Sciences
                0027-8424
                1091-6490
                September 24 2013
                August 12 2013
                September 24 2013
                : 110
                : 39
                : 15644-15649
                Affiliations
                [1 ]Morgridge Institute for Research, Madison, WI 53715;
                [2 ]Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208-3500;
                [3 ]Department of Cell and Regenerative Biology, University of Wisconsin–Madison, Madison, WI 53706; and
                [4 ]Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106
                Article
                10.1073/pnas.1313587110
                3785731
                23940360
                2177ce6f-5660-41a1-9a6a-9bb1b701d19d
                © 2013
                History

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