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      Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach

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          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

          We develop an in vivo library-on-library methodology to simultaneously assess single guide RNA (sgRNA) activity across ~1,400 genomic loci. Assaying across multiple human cell types, end-processing enzymes, and two Cas9 orthologs, we unravel underlying nucleotide sequence and epigenetic parameters. Our results enable improved design of reagents, shed light on mechanisms of genome targeting, and provide a generalizable framework to study nucleic acid-nucleic acid interactions and biochemistry in high throughput.

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          An Integrated Encyclopedia of DNA Elements in the Human Genome

          Iakes Ezkurdia (2016)
          Summary The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure, and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall the project provides new insights into the organization and regulation of our genes and genome, and an expansive resource of functional annotations for biomedical research.
          Article 0 comments Cited 3414 times – based on 0 reviews     Review now
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            BigWig and BigBed: enabling browsing of large distributed datasets

            W. J. Kent, A. S. Zweig, Lisa Barber … (2010)
            Summary: BigWig and BigBed files are compressed binary indexed files containing data at several resolutions that allow the high-performance display of next-generation sequencing experiment results in the UCSC Genome Browser. The visualization is implemented using a multi-layered software approach that takes advantage of specific capabilities of web-based protocols and Linux and UNIX operating systems files, R trees and various indexing and compression tricks. As a result, only the data needed to support the current browser view is transmitted rather than the entire file, enabling fast remote access to large distributed data sets. Availability and implementation: Binaries for the BigWig and BigBed creation and parsing utilities may be downloaded at http://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/. Source code for the creation and visualization software is freely available for non-commercial use at http://hgdownload.cse.ucsc.edu/admin/jksrc.zip, implemented in C and supported on Linux. The UCSC Genome Browser is available at http://genome.ucsc.edu Contact: ann@soe.ucsc.edu Supplementary information: Supplementary byte-level details of the BigWig and BigBed file formats are available at Bioinformatics online. For an in-depth description of UCSC data file formats and custom tracks, see http://genome.ucsc.edu/FAQ/FAQformat.html and http://genome.ucsc.edu/goldenPath/help/hgTracksHelp.html
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              Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.

              Cem Kuscu, Sevki Arslan, Ritambhara Singh … (2014)
              RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ∼10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide.
              Article 0 comments Cited 327 times – based on 0 reviews     Review now
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                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: 10 January 2017
                Publication date (Electronic): 13 July 2015
                Publication date (Print): September 2015
                Publication date PMC-release: 06 February 2017
                Volume: 12
                Issue: 9
                Pages: 823-826
                Affiliations
                [1 ]Department of Genetics, Harvard Medical School, Boston, MA, USA
                [2 ]Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
                [3 ]Scripps Institute of Oceanography, University of California San Diego, La Jolla, CA, USA
                [4 ]Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
                Author notes
                [† ]Correspondence can be addressed to: Prashant Mali, pmali@ 123456ucsd.edu , George Church, gchurch@ 123456genetics.med.harvard.edu
                [5]

                These authors contributed equally

                Article
                Manuscript ID: NIHMS700024
                DOI: 10.1038/nmeth.3473
                PMC ID: 5292764
                PubMed ID: 26167643
                SO-VID: 3bcabab9-e5e1-4be1-84af-85868d789725
                License:

                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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                ScienceOpen disciplines: Life sciences

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