Abstract:
:Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of target site recognition. Cleavage specificity is typically evaluated by low throughput assays (T7 endonuclease I assay, target amplification followed by high-throughput sequencing), which are limited to a subset of potential off-target sites. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites for two guide RNAs. RNA-guided Cas9 binding was highly specific to the target site while off-target binding occurred at much lower intensities. Cas9-bound regions were highly enriched in NGG sites, a sequence required for target site recognition by Streptococcus pyogenes Cas9. To determine the relationship between Cas9 binding and endonuclease activity, we applied targeted sequence capture, which allowed us to survey 1200 genomic loci simultaneously including potential off-target sites identified by ChIP-seq and by computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results confirm the high-specificity of CRISPR endonucleases and demonstrate that sequence capture can be used as a high-throughput genome-wide approach to identify off-target activity.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
O'Geen H,Henry IM,Bhakta MS,Meckler JF,Segal DJdoi
10.1093/nar/gkv137subject
Has Abstractpub_date
2015-03-31 00:00:00pages
3389-404issue
6eissn
0305-1048issn
1362-4962pii
gkv137journal_volume
43pub_type
杂志文章abstract::The Mouse Genome Database (MGD) forms the core of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a model organism database resource for the laboratory mouse. MGD provides essential integration of experimental knowledge for the mouse system with information annotated from both literature an...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gki113
更新日期:2005-01-01 00:00:00
abstract::We have compiled the DNA sequence data for E. coli available from the GENBANK and EMBL data libraries and over a period of several years independently from the literature. This is the fifth listing replacing and increasing the former listings substantially. However, in order to save space this printed version contains...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/21.13.2973
更新日期:1993-07-01 00:00:00
abstract::Plasmids containing a dihydrofolate reductase (DHFR) expression unit were transfected into DHFR-deficient Chinese hamster ovary (CHO) cells. Methotrexate exposure was used to select cells with amplified DHFR sequences. Three cell lines were isolated containing amplified copies of transfected DNA that had integrated in...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/17.4.1697
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abstract::A DNA fragment d(GCGAAAGCT), known to adopt a stable mini-hairpin structure in solution, has been crystallized in the space group I4(1)22 with the unit-cell dimensions a = b = 53.4 A and c = 54.0 A, and the crystal structure has been determined at 2.5 A resolution. The four nucleotide residues CGAA of the first half o...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkf639
更新日期:2002-12-01 00:00:00
abstract::Although cis-regulatory binding sites (CRBSs) are at least as important as the coding sequences in a genome, our general understanding of them in most sequenced genomes is very limited due to the lack of efficient and accurate experimental and computational methods for their characterization, which has largely hindere...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkp248
更新日期:2009-06-01 00:00:00
abstract::p73, the p53 homologue, exists as a transactivation-domain-proficient TAp73 or deficient deltaN(DN)p73 form. Expectedly, the oncogenic DNp73 that is capable of inactivating both TAp73 and p53 function, is over-expressed in cancers. However, the role of TAp73, which exhibits tumour-suppressive properties in gain or los...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkn414
更新日期:2008-08-01 00:00:00
abstract::The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5' end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We r...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa789
更新日期:2020-10-09 00:00:00
abstract::The nucleotide sequence of the promoter region and the first five genes of the atp (or unc) operon of Escherichia coli has been determined. The first proposed gene in the operon contains four AUA codons and may be poorly expressed; it encodes a basic but yet hydrophobic protein which could function as a pilot protein ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/9.16.3919
更新日期:1981-08-25 00:00:00
abstract::A 5',7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and a textbook aspect of co-transcriptional RNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). Interest in the CBC has recently renewed...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa743
更新日期:2020-10-09 00:00:00
abstract::7,8-Dihydro-8-oxo-deoxyguanosine (8oxodG) is a highly premutagenic DNA lesion due to its ability to mispair with adenine. Schizosaccharomyces pombe lacks homologs for relevant enzymes that repair 8oxodG, which suggests that this lesion could be persistent and must be tolerated. Here we show that SpPol4, the unique Pol...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gku711
更新日期:2014-09-01 00:00:00
abstract::PvuRts1I is a prototype for a larger family of restriction endonucleases that cleave DNA containing 5-hydroxymethylcytosine (5hmC) or 5-glucosylhydroxymethylcytosine (5ghmC), but not 5-methylcytosine (5mC) or cytosine. Here, we report a crystal structure of the enzyme at 2.35 Å resolution. Although the protein has bee...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gku186
更新日期:2014-05-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa915
更新日期:2021-01-08 00:00:00
abstract::The Escherichia coli fmu gene product has recently been determined to be the 16S rRNA m(5)C 967 methyltransferase. As such, Fmu represents the first protein identified as an S -adenosyl-L-methionine (AdoMet)- dependent RNA m(5)C methyltransferase whose amino acid sequence is known. Using the amino acid sequence of Fmu...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/27.15.3138
更新日期:1999-08-01 00:00:00
abstract::An oligodeoxyribonucleotide, d(GCTCACAAT-X-ATTGTGAGC), where X represents a hexaethylene glycol chain, was studied using circular dichroism spectroscopy. Its conformation and conformational stability were compared to those of compounds where X was replaced by four thymines and to the duplex of same sequence without lo...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/18.21.6353
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abstract::The inducibility of the vir genes (virA, -B, -C, -D, -E, and -G) on pRiA4 was examined at the transcriptional level, and the RNA-starting sites were determined by S1-nuclease mapping and primer-extension experiments. All of these genes were inducible, while virA, -E, and -G were transcribed even under noninducing cond...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/17.21.8711
更新日期:1989-11-11 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkz286
更新日期:2019-06-20 00:00:00
abstract::Motor proteins that translocate on nucleic acids are key players in gene expression and maintenance. While the function of these proteins is diverse, they are driven by highly conserved core motor domains. In transcription-coupled DNA repair, motor activity serves to remove RNA polymerase stalled on damaged DNA, makin...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkp680
更新日期:2009-10-01 00:00:00
abstract::The major mRNA degradation pathway involves deadenylation of the target molecule followed by decapping and, finally, 5'-->3' exonuclease digestion of the mRNA body. While yeast factors involved in the decapping and exonuclease degradation steps have been identified, the nature of the factor(s) involved in the deadenyl...
journal_title:Nucleic acids research
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky378
更新日期:2018-07-02 00:00:00
abstract::The KEGG RPAIR database is a collection of biochemical structure transformation patterns, called RDM patterns, and chemical structure alignments of substrate-product pairs (reactant pairs) in all known enzyme-catalyzed reactions taken from the Enzyme Nomenclature and the KEGG PATHWAY database. Here, we present PathPre...
journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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doi:10.1093/nar/20.11.2835
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkm088
更新日期:2007-01-01 00:00:00
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journal_title:Nucleic acids research
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doi:10.1093/nar/24.11.2087
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkr914
更新日期:2012-01-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkz293
更新日期:2019-07-09 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/15.20.8439
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abstract::The diverse localization of transcripts in cells suggests that there are many specific RNA-protein interactions that have yet to be identified. Progress has been limited, however, by the lack of a robust method to detect and isolate the RNA-binding proteins. Here we describe the use of an RNA aptamer, scaffolded to a ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkq1316
更新日期:2011-04-01 00:00:00
abstract::Adenosine-to-inosine (A-to-I) RNA editing is a common post transcriptional modification. It has a critical role in protecting against false activation of innate immunity by endogenous double stranded RNAs and has been associated with various regulatory processes and diseases such as autoimmune and cardiovascular disea...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa305
更新日期:2020-06-19 00:00:00