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 Zhang101
Estimated H-index: 101
Abstract
Analyses of the determinants of the specificity of Cas9 nuclease provide rules for selecting optimal target sites.
  • References (27)
  • Citations (1894)
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References27
Published on Jan 1, 2010in Methods of Molecular Biology
Dmitry Guschin27
Estimated H-index: 27
(Sangamo BioSciences),
Adam James Waite7
Estimated H-index: 7
(Sangamo BioSciences)
+ 3 AuthorsEdward J. Rebar42
Estimated H-index: 42
(Sangamo BioSciences)
The development of zinc finger nucleases for targeted gene modification can benefit from rapid functional assays that directly quantify activity at the endogenous target. Here we describe a simple procedure for quantifying mutations that result fromDNA double-strand break repair via non-homologous end joining. The assay is based on the ability of the Surveyor nuclease to selectively cleave distorted duplex DNA formed via cross-annealing of mutated and wild-type sequence.
313 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 Joung59
Estimated H-index: 59
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...
1,571 Citations Source Cite
Magdy M. Mahfouz24
Estimated H-index: 24
,
Lixin Li9
Estimated H-index: 9
+ 3 AuthorsJian-Kang Zhu134
Estimated H-index: 134
Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem r...
337 Citations Source Cite
Published on Mar 1, 2013in Nature Biotechnology 35.72
Wenyan Jiang11
Estimated H-index: 11
,
David Bikard17
Estimated H-index: 17
+ 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.
1,160 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...
487 Citations Source Cite
Published on Feb 15, 2013in Science 41.06
Prashant Mali28
Estimated H-index: 28
(Harvard University),
Luhan Yang16
Estimated H-index: 16
(Harvard University)
+ 5 AuthorsGeorge M Church G M132
Estimated H-index: 132
(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...
4,240 Citations Source Cite
Published on Jan 1, 2012in Nature Protocols 12.42
Neville E. Sanjana20
Estimated H-index: 20
(McGovern Institute for Brain Research),
Le Cong15
Estimated H-index: 15
(McGovern Institute for Brain Research)
+ 3 AuthorsFeng Zhang101
Estimated H-index: 101
(McGovern Institute for Brain Research)
438 Citations Source Cite
Published on Apr 1, 2011in Methods 4.00
Sylwia Bobis-Wozowicz1
Estimated H-index: 1
(Hannover Medical School),
Anna Osiak1
Estimated H-index: 1
(Hannover Medical School)
+ 1 AuthorsToni Cathomen37
Estimated H-index: 37
(Hannover Medical School)
Abstract Zinc-finger nucleases (ZFNs) are designer nucleases capable of cleaving a prespecified target DNA within complex genomes. ZFNs consist of a non-specific endonuclease domain fused to an engineered DNA-binding domain that tethers the nuclease activity to the chosen chromosomal site. The endonuclease-induced DNA double strand break triggers a cellular DNA damage response, resulting in double strand break repair by either accurate homologous recombination (HR) or error-prone non-homologous ...
35 Citations Source Cite
Published on Apr 1, 1981in Cell 31.40
Daniel F. Bogenhagen4
Estimated H-index: 4
(Carnegie Institution for Science),
Donald D. Brown67
Estimated H-index: 67
(Carnegie Institution for Science)
Abstract We have analyzed the DNA sequences required for termination of Xenopus 5S RNA synthesis in vitro. Termination occurs within clusters of four or more consecutive T residues in the noncoding DNA strand sequence. The 3′ flanking sequence following the gene is not required for termination. The distance between the T cluster and the site of transcription initiation as well as the exact nucleotide sequences preceding the T cluster can be varied without impairing termination. Thus the sequence...
280 Citations Source Cite
Published on Feb 1, 2011in Nature Biotechnology 35.72
Jeffrey C. Miller35
Estimated H-index: 35
(Sangamo BioSciences),
Siyuan Tan4
Estimated H-index: 4
(Sangamo BioSciences)
+ 17 AuthorsSarah J. Hinkley6
Estimated H-index: 6
(Sangamo BioSciences)
TALEs (transcription activator-like effectors) are transcription factors from the plant pathogen Xanthomonas that can be readily engineered to bind new DNA sequences of interest. Miller et al. use a truncated TALE linked to a nuclease domain to edit and regulate endogenous genes in human cells.
1,387 Citations Source Cite
  • References (27)
  • Citations (1894)
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Cited By1894
Published on Jan 1, 2014in Methods in Enzymology 1.98
Zengrong Zhu6
Estimated H-index: 6
(Kettering University),
Federico González6
Estimated H-index: 6
(Kettering University),
Danwei Huangfu20
Estimated H-index: 20
(Kettering University)
Abstract Human pluripotent stem cells (hPSCs) have the potential to generate all adult cell types, including rare or inaccessible human cell populations, thus providing a unique platform for disease studies. To realize this promise, it is essential to develop methods for efficient genetic manipulations in hPSCs. Established using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, the iCRIS...
30 Citations Source Cite
Published on Jan 1, 2014in Methods in Enzymology 1.98
Benjamin L. Oakes8
Estimated H-index: 8
(University of California, Berkeley),
Dana C. Nadler5
Estimated H-index: 5
(University of California, Berkeley),
David F. Savage19
Estimated H-index: 19
(University of California, Berkeley)
Abstract CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complementary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of...
8 Citations Source Cite
Published on Sep 1, 2014in BioTechniques 2.10
Qiupeng Zheng5
Estimated H-index: 5
,
Xiaohong Cai1
Estimated H-index: 1
+ 5 AuthorsShenglin Huang27
Estimated H-index: 27
The prokaryotic type II CRISPR/Cas9 system has been adapted to perform targeted genome editing in cells and model organisms. Here, we describe targeted gene deletion and replacement in human cells via the CRISPR/Cas9 system using two guide RNAs. The system effectively generated targeted deletions of varied length, regardless of the transcriptional status of the target gene. It is notable that targeted gene deletions generated via CRISPR/Cas9 and two guide RNAs resulted in the formation of correc...
55 Citations Source Cite
Published on Nov 1, 2013in Hereditas 1.63
J. Li33
Estimated H-index: 33
(Chinese Academy of Sciences),
Chen Kl1
Estimated H-index: 1
+ 4 AuthorsGao Cx1
Estimated H-index: 1
Bacteria and archaea have evolved an adaptive immune system, known as typeⅡprokaryotic clustered regu- larly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, which uses short RNA to direct the degradation of target sequences present in invading viral and plasmid DNAs. Recent advances in CRISPR/Cas system pro- vide an improved method for genome editing, showing robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci. It is the latest ...
6 Citations
Published on Sep 1, 2015in Hearing Research 2.82
Bing Zou4
Estimated H-index: 4
(University of Miami),
Rahul Mittal11
Estimated H-index: 11
(University of Miami)
+ 9 AuthorsShiming Yang13
Estimated H-index: 13
(Chinese PLA General Hospital)
Abstract Targeted genome editing mediated by clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) technology has emerged as one of the most powerful tools to study gene functions, and with potential to treat genetic disorders. Hearing loss is one of the most common sensory disorders, affecting approximately 1 in 500 newborns with no treatment. Mutations of inner ear genes contribute to the largest portion of genetic deafness. The simplicity and ...
17 Citations Source Cite
Published on Jan 1, 2014in Methods in Enzymology 1.98
Owen W. Ryan3
Estimated H-index: 3
(University of California, Berkeley),
Jamie H. D. Cate50
Estimated H-index: 50
Abstract Global demand has driven the use of industrial strains of the yeast Saccharomyces cerevisiae for large-scale production of biofuels and renewable chemicals. However, the genetic basis of desired domestication traits is poorly understood because robust genetic tools do not exist for industrial hosts. We present an efficient, marker-free, high-throughput, and multiplexed genome editing platform for industrial strains of S. cerevisiae that uses plasmid-based expression of the CRISPR/Cas9 e...
32 Citations Source Cite
Published on Jan 1, 2014in Methods in Enzymology 1.98
Carolin Anders8
Estimated H-index: 8
(University of Zurich),
Martin Jinek25
Estimated H-index: 25
(University of Zurich)
Cas9 is a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNAs with complementarity to the guide RNA. The programmable sequence specificity of Cas9 has been harnessed for genome editing and gene expression control in many organisms. Here, we describe protocols for the heterologous expression and purification of recombinant Cas9 protein and for in vitro transcription of guide RNAs. We describe in vitro reconstitution of the Cas9-guide RNA ribonucleoprotein ...
20 Citations Source Cite
Published on Jan 1, 2015in Advances in Experimental Medicine and Biology 1.76
Tatjana I. Cornu1
Estimated H-index: 1
(University Medical Center Freiburg),
Claudio Mussolino15
Estimated H-index: 15
(University Medical Center Freiburg)
+ 1 AuthorsToni Cathomen37
Estimated H-index: 37
(University Medical Center Freiburg)
Acquired immunodeficiency syndrome (AIDS) is a life-threatening disorder caused by infection of individuals with the human immunodeficiency virus (HIV). Entry of HIV-1 into target cells depends on the presence of two surface proteins on the cell membrane: CD4, which serves as the main receptor, and either CCR5 or CXCR4 as a co-receptor. A limited number of people harbor a genomic 32-bp deletion in the CCR5 gene (CCR5∆32), leading to expression of a truncated gene product that provides resistance...
11 Citations Source Cite
Published on Aug 1, 2015in Journal of Biotechnology 2.53
Xiquan Liang3
Estimated H-index: 3
(Thermo Fisher Scientific),
Jason Potter4
Estimated H-index: 4
(Thermo Fisher Scientific)
+ 9 AuthorsSridhar Ranganathan6
Estimated H-index: 6
(Thermo Fisher Scientific)
CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation...
183 Citations Source Cite
Published on May 28, 2015in Journal of Visualized Experiments 1.18
Shenglan Li6
Estimated H-index: 6
(University of Texas Health Science Center at Houston),
Haipeng Xue21
Estimated H-index: 21
(University of Texas Health Science Center at Houston)
+ 3 AuthorsYun Liu43
Estimated H-index: 43
(University of Texas Health Science Center at Houston)
Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript desc...
3 Citations Source Cite