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DNA targeting specificity of RNA-guided Cas9 nucleases

Published on Sep 1, 2013in Nature Biotechnology 35.72
· DOI :10.1038/nbt.2647
Patrick Hsu15
Estimated H-index: 15
,
Douglas Scott96
Estimated H-index: 96
+ 11 AuthorsFeng Zhang104
Estimated H-index: 104
Abstract
Analyses of the determinants of the specificity of Cas9 nuclease provide rules for selecting optimal target sites.
  • References (28)
  • Citations (2006)
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References28
Newest
Published on Aug 1, 2013in Genetics 4.08
Scott J. Gratz9
Estimated H-index: 9
(University of Wisconsin-Madison),
Alexander M. Cummings3
Estimated H-index: 3
(University of Wisconsin-Madison)
+ 5 AuthorsKate M. O’Connor-Giles6
Estimated H-index: 6
(University of Wisconsin-Madison)
We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.
539 Citations Source Cite
Published on May 1, 2013in Cell 31.40
Haoyi Wang24
Estimated H-index: 24
(Massachusetts Institute of Technology),
Hui Yang16
Estimated H-index: 16
(Massachusetts Institute of Technology)
+ 4 AuthorsRudolf Jaenisch173
Estimated H-index: 173
(Massachusetts Institute of Technology)
SUMMARY Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cell...
2,009 Citations Source Cite
Published on May 1, 2013in Cell Research 15.39
Bin Shen14
Estimated H-index: 14
,
Jun Zhang3
Estimated H-index: 3
+ 6 AuthorsXingxu Huang14
Estimated H-index: 14
374 Citations Source Cite
Published on Apr 1, 2013in Cell Research 15.39
Nannan Chang8
Estimated H-index: 8
,
Changhong Sun1
Estimated H-index: 1
+ 5 AuthorsJianzhong Jeff Xi1
Estimated H-index: 1
Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site- specific somatic mutations were found when specif...
500 Citations Source Cite
Published on Mar 1, 2013in Nature Biotechnology 35.72
Wenyan Jiang11
Estimated H-index: 11
,
David Bikard18
Estimated H-index: 18
+ 2 AuthorsLuciano A. Marraffini33
Estimated H-index: 33
A CRISPR-Cas system is harnessed to introduce template-driven mutations in S. pneumoniae and E. coli at high efficiency without requiring selectable markers.
1,222 Citations Source Cite
Published on Mar 1, 2013in Nature Biotechnology 35.72
Woong Y. Hwang5
Estimated H-index: 5
,
Yanfang Fu14
Estimated H-index: 14
+ 6 AuthorsJ. Keith Joung60
Estimated H-index: 60
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 zebra...
1,633 Citations Source Cite
Published on Mar 1, 2013in Nature Biotechnology 35.72
Seung Woo Cho25
Estimated H-index: 25
,
Sojung C. Kim1
Estimated H-index: 1
+ 1 AuthorsJin-Soo Kim52
Estimated H-index: 52
Abstract We employ the CRISPR-Cas system of Streptococcus pyogenes as programmable RNA-guided endonucleases (RGENs) to cleave DNA in a targeted manner for genome editing in human cells. We show that complexes of the Cas9 protein and artificial chimeric RNAs efficiently cleave two genomic sites and induce indels with frequencies of up to 33%.
1,078 Citations Source Cite
Published on Feb 15, 2013in Science 41.06
Le Cong15
Estimated H-index: 15
(Massachusetts Institute of Technology),
F. Ann Ran9
Estimated H-index: 9
(Massachusetts Institute of Technology)
+ 8 AuthorsLuciano A. Marraffini33
Estimated H-index: 33
(Rockefeller University)
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...
6,233 Citations Source Cite
Published on Feb 15, 2013in Science 41.06
Prashant Mali29
Estimated H-index: 29
(Harvard University),
Luhan Yang15
Estimated H-index: 15
(Harvard University)
+ 5 AuthorsGeorge M Church G M135
Estimated H-index: 135
(Wyss Institute for Biologically Inspired Engineering)
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 cel...
4,460 Citations Source Cite
Published on Feb 1, 2013in Cell 31.40
Lei S. Qi28
Estimated H-index: 28
(University of California, San Francisco),
Matthew H. Larson12
Estimated H-index: 12
(University of California, San Francisco)
+ 4 AuthorsWendell A. Lim72
Estimated H-index: 72
SUMMARY Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase b...
1,750 Citations Source Cite
Cited By2006
Newest
Published on Jan 25, 2019in Genome Biology 13.21
Jean-Philippe Fortin9
Estimated H-index: 9
(Genentech),
Jenille Tan5
Estimated H-index: 5
(Genentech)
+ 4 AuthorsScott E. Martin4
Estimated H-index: 4
(Genentech)
Background Genome-wide loss-of-function screens using the CRISPR/Cas9 system allow the efficient discovery of cancer cell vulnerabilities. While several studies have focused on correcting for DNA cleavage toxicity biases associated with copy number alterations, the effects of sgRNAs co-targeting multiple genomic loci in CRISPR screens have not been discussed.
4 Citations Source Cite
Published on Mar 8, 2019in Nature Communications 12.35
Grégoire Cullot (French Institute of Health and Medical Research), Julian Boutin (French Institute of Health and Medical Research)+ 17 AuthorsVéronique Guyonnet-Duperat12
Estimated H-index: 12
(French Institute of Health and Medical Research)
CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-de...
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Published on Jan 8, 2019
D. Ghosh , P. Venkataramani + 1 AuthorsSonali Bhattacharjee5
Estimated H-index: 5
Genome editing allows for the precise manipulation of DNA sequences in a cell making this technology essential for understanding gene function. CRISPR/Cas9 is a targeted genome-editing platform derived from bacterial adaptive immune system and has been repurposed into a genome-editing tool. The RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, making this technology easier, more efficient, scalable and an indispensable tool in biol...
Published on May 3, 2019
Jie Qiao1
Estimated H-index: 1
(Hubei University),
wenqiang li1
Estimated H-index: 1
(Hubei University)
+ 3 AuthorsYi Liu1
Estimated H-index: 1
(Hubei University)
CRISPR/Cas9 ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications. However, to date there are no efficient methods that can produce these proteins at large scales and low cost. Here, we present a streamlined method for direct production of Cas9 RNPs from Escherichia coli by co-expression of Cas9 and the target-specific single-guided RNAs. Harnessing an ultrahigh-affinity CL7/Im7 purification system recently developed we achieve one-step purificatio...
1 Citations Source Cite
Published on Jan 28, 2019in BMC Biotechnology 2.60
Background Recent innovation in the field of genome engineering encompasses numerous levels of plant genome engineering which attract the substantial excitement of plant biologist worldwide. RNA-guided CRISPR Cas9 system has appeared a promising tool in site-directed mutagenesis due to its innovative utilization in different branches of biology. CRISPR-Cas9 nuclease system have supersedes all previously existed strategies and their associated pitfalls encountered with site-specific mutagenesis.
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Published on Mar 5, 2019in Nature Communications 12.35
Yueping Zhang1
Estimated H-index: 1
(Beijing University of Chemical Technology),
Juan Wang1
Estimated H-index: 1
(Beijing University of Chemical Technology)
+ 4 AuthorsZihe Liu15
Estimated H-index: 15
(Beijing University of Chemical Technology)
With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. Here, we report a gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering of Saccharomyces cerevisiae. Using reported gRNAs shown to be effective, this system enables simultaneous disruption of 8 genes with 87% efficiency. We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming th...
1 Citations Source Cite
Published on Apr 8, 2019in Nature Communications 12.35
Lapatrada Taemaitree (University of Oxford), Arun Shivalingam2
Estimated H-index: 2
(University of Oxford)
+ 1 AuthorsTom Brown61
Estimated H-index: 61
(University of Oxford)
As the applications of CRISPR-Cas9 technology diversify and spread beyond the laboratory to diagnostic and therapeutic use, the demands of gRNA synthesis have increased and access to tailored gRNAs is now restrictive. Enzymatic routes are time-consuming, difficult to scale-up and suffer from polymerase-bias while existing chemical routes are inefficient. Here, we describe a split-and-click convergent chemical route to individual or pools of sgRNAs. The synthetic burden is reduced by splitting th...
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Published on Mar 7, 2019in Nature Communications 12.35
Victor Heurtier1
Estimated H-index: 1
(Pasteur Institute),
Nick Owens3
Estimated H-index: 3
(Pasteur Institute)
+ 6 AuthorsPablo Navarro16
Estimated H-index: 16
(Pasteur Institute)
Transcription factor networks, together with histone modifications and signalling pathways, underlie the establishment and maintenance of gene regulatory architectures associated with the molecular identity of each cell type. However, how master transcription factors individually impact the epigenomic landscape and orchestrate the behaviour of regulatory networks under different environmental constraints is only partially understood. Here, we show that the transcription factor Nanog deploys mult...
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Published on Jan 25, 2019in Nature Communications 12.35
Junjie Tan1
Estimated H-index: 1
(Max Planck Society),
Fei Zhang1
Estimated H-index: 1
(Max Planck Society)
+ 1 AuthorsRalph Bock62
Estimated H-index: 62
(Max Planck Society)
RNA-guided nucleases of the CRISPR/Cas type can be repurposed as programmable nucleotide deaminases to mediate targeted nucleotide substitutions. Such base editors have enormous potential in genome editing, gene therapy and precision breeding. However, current editors suffer from limited specificity in that they edit different and/or multiple bases within a larger sequence window. Using cytidine deaminase base editors that elicit C-to-T mutations, we show here that high editing precision can be ...
1 Citations Source Cite