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      Engineered CRISPR-Cas9 nucleases with altered PAM specificities

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          Abstract

          Although CRISPR-Cas9 nucleases are widely used for genome editing 1, 2 , the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif ( PAM) 36 . As a result, it can often be difficult to target double-stranded breaks ( DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 ( SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-Seq analysis 7 . In addition, we identified and characterized another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also found that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 ( St1Cas9) and Staphylococcus aureus Cas9 ( SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

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          Most cited references 14

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          Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms.
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            Phage response to CRISPR-encoded resistance in Streptococcus thermophilus.

            Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.
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              FLASH Assembly of TALENs Enables High-Throughput Genome Editing

              Engineered transcription activator-like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) platform, a rapid and cost-effective method we developed to enable large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell-based EGFP reporter system and found that all 48 possessed efficient gene modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with engineered zinc-finger nucleases or meganucleases.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                12 August 2015
                22 June 2015
                23 July 2015
                23 January 2016
                : 523
                : 7561
                : 481-485
                Affiliations
                [1 ]Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129 USA
                [2 ]Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA 02129 USA
                [3 ]Department of Pathology, Harvard Medical School, Boston, MA 02115 USA
                [4 ]Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden
                [5 ]Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129 USA
                [6 ]Department of Systems Biology, Harvard Medical School, Boston, MA 02115 USA
                [7 ]Broad Institute, Cambridge, MA 02142 USA
                Author notes
                Correspondence and requests for materials should be addressed to jjoung@ 123456mgh.harvard.edu
                Article
                NIHMS696684
                10.1038/nature14592
                4540238
                26098369

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