For Publishers
DiscoveryMetadataPeer reviewHostingPublishing
For Researchers
JoinPublishReviewCollect
Register
Blog
About
Search
Advanced search
My ScienceOpen
Search
For Publishers
For Researchers
Blog
About
378
views
10
references
 Top references
cited by
318
    
0 reviews
 Review
0
comments
 Comment
0
recommends
+1 Recommend
0 collections
    0
    shares
      422
      similar
       All similar
      • Record: found
      • Abstract: found
      • Article: not found

      Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences and identify a highly targetable protein from Neisseria meningitidis. Our results provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins and adapting them to eukaryotic cells.

          Related collections

          Most cited references10

          • Record: found
          • Abstract: found
          • Article: not found

          JCat: a novel tool to adapt codon usage of a target gene to its potential expression host

          Andreas Gerber Grote, Karsten Hiller, Maurice Scheer … (2005)
          A novel method for the adaptation of target gene codon usage to most sequenced prokaryotes and selected eukaryotic gene expression hosts was developed to improve heterologous protein production. In contrast to existing tools, JCat (Java Codon Adaptation Tool) does not require the manual definition of highly expressed genes and is, therefore, a very rapid and easy method. Further options of JCat for codon adaptation include the avoidance of unwanted cleavage sites for restriction enzymes and Rho-independent transcription terminators. The output of JCat is both graphically and as Codon Adaptation Index (CAI) values given for the pasted sequence and the newly adapted sequence. Additionally, a list of genes in FASTA-format can be uploaded to calculate CAI values. In one example, all genes of the genome of Caenorhabditis elegans were adapted to Escherichia coli codon usage and further optimized to avoid commonly used restriction sites. In a second example, the Pseudomonas aeruginosa exbD gene codon usage was adapted to E.coli codon usage with parallel avoidance of the same restriction sites. For both, the degree of introduced changes was documented and evaluated. JCat is integrated into the PRODORIC database that hosts all required information on the various organisms to fulfill the requested calculations. JCat is freely accessible at .
          Article 0 comments Cited 496 times – based on 0 reviews     Review now
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus.

            Philippe Horvath, Dennis A Romero, Anne-Claire Coûté-Monvoisin … (2008)
            Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes that provide acquired immunity against foreign genetic elements. Here, we characterize a novel Streptococcus thermophilus locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several S. thermophilus strains. We show that both CRISPR repeats and cas genes are locus specific and functionally coupled. A total of 124 strains were studied, and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique), and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%), and S. thermophilus chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers, cas genes, CRISPR motif, locus architecture, and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers after exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that the activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the coevolution of host and viral genomes.
            Article 0 comments Cited 283 times – based on 0 reviews     Review now
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.

              Y G Kim, J. Cha, S Chandrasegaran (1996)
              A long-term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create "artificial" nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.
              Article 0 comments Cited 271 times – based on 0 reviews     Review now
                Bookmark
                All references

                Author and article information

                Journal
                Journal ID (nlm-journal-id): 101215604
                Journal ID (pubmed-jr-id): 32338
                Journal ID (nlm-ta): Nat Methods
                Journal ID (iso-abbrev): Nat. Methods
                Title: Nature methods
                ISSN (Print): 1548-7091
                ISSN (Electronic): 1548-7105
                Publication date Nihms-submitted: 9 November 2013
                Publication date (Electronic): 29 September 2013
                Publication date (Print): November 2013
                Publication date PMC-release: 01 May 2014
                Volume: 10
                Issue: 11
                Electronic Location Identifier: 10.1038/nmeth.2681
                Affiliations
                [1 ]Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA 02115, USA
                [2 ]Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
                [3 ]Program in Medical Engineering & Medical Physics, Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
                Author notes
                [5 ]Correspondence should be addressed to: gchurch@ 123456genetics.med.harvard.edu
                [4]

                These authors contributed equally to this work.

                Article
                Manuscript ID: NIHMS522590
                DOI: 10.1038/nmeth.2681
                PMC ID: 3844869
                PubMed ID: 24076762
                SO-VID: 3d689f0d-d63f-4b9e-a852-a913475bccd3
                License:

                Users may view, print, copy, download and 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

                History
                Funding
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: P50 HG005550 || HG
                Categories
                Subject: Article

                ScienceOpen disciplines: Life sciences
                Data availability:
                ScienceOpen disciplines: Life sciences

                Comments

                Comment on this article

                scite_

                Similar content422

                See all similar

                Cited by310

                See all cited by

                Most referenced authors959

                See all reference authors