RNA-Guided Human Genome Engineering via Cas9

Published on Feb 15, 2013in Science 41.06
· DOI :10.1126/science.1232033
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)
Abstract
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 cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
  • References (46)
  • Citations (4240)
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References46
Published on Sep 1, 2012in Nature 41.58
Robert E. Thurman34
Estimated H-index: 34
(University of Washington),
Eric Rynes8
Estimated H-index: 8
(University of Washington)
+ 59 AuthorsBenjamin Vernot19
Estimated H-index: 19
(University of Washington)
An extensive map of human DNase I hypersensitive sites, markers of regulatory DNA, in 125 diverse cell and tissue types is described; integration of this information with other ENCODE-generated data sets identifies new relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns.
1,334 Citations Source Cite
Published on Jul 1, 2009in Cell Stem Cell 23.29
Jizhong Zou11
Estimated H-index: 11
(Johns Hopkins University School of Medicine),
Morgan L. Maeder26
Estimated H-index: 26
(Harvard University)
+ 10 AuthorsGeorge Q. Daley105
Estimated H-index: 105
(Harvard University)
Summary We report here homologous recombination (HR)-mediated gene targeting of two different genes in human iPS cells (hiPSCs) and human ES cells (hESCs). HR-mediated correction of a chromosomally integrated mutant GFP reporter gene reaches efficiencies of 0.14%–0.24% in both cell types transfected by donor DNA with plasmids expressing zinc finger nucleases (ZFNs). Engineered ZFNs that induce a sequence-specific double-strand break in the GFP gene enhanced HR-mediated correction by > 1400-fold ...
440 Citations Source Cite
Published on Nov 1, 2010in Nature 41.58
Josiane E. Garneau6
Estimated H-index: 6
(Laval University),
Marie-Ève Dupuis4
Estimated H-index: 4
(Laval University)
+ 7 AuthorsSylvain Moineau50
Estimated H-index: 50
(Laval University)
CRISPR/Cas is a microbial immune system that is known to protect bacteria from virus infection. These authors show that the Streptococcus thermophilus CRISPR/Cas system can prevent both plasmid carriage and phage infection through cleavage of invading double-stranded DNA.
935 Citations Source Cite
Published on Dec 11, 2009in Science 41.06
Jens Boch30
Estimated H-index: 30
,
Heidi Scholze6
Estimated H-index: 6
+ 6 AuthorsUlla Bonas58
Estimated H-index: 58
Xanthomonas bacteria attack their plant hosts by delivering their own transcription-activator–like (TAL) proteins into the plant cell nucleus and alter the plant's gene regulation (see the Perspective by [ Voytas and Joung ][1] ). Moscou and Bogdanove (p. [1501][2], published online 29 October: see the cover) and Boch et al. (p. [1509][3], published online 29 October) have now discovered how the similar but not identical repeats in the TAL proteins encode the specificity needed for the proteins ...
1,591 Citations Source Cite
Published on Nov 1, 2007in Nature Biotechnology 35.72
Angelo Lombardo19
Estimated H-index: 19
(Vita-Salute San Raffaele University),
Pietro Genovese10
Estimated H-index: 10
(Vita-Salute San Raffaele University)
+ 9 AuthorsPhilip D. Gregory61
Estimated H-index: 61
(Sangamo BioSciences)
Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery
726 Citations Source Cite
Published on Oct 27, 2011in Blood 15.13
Jizhong Zou11
Estimated H-index: 11
,
Prashant Mali28
Estimated H-index: 28
(Johns Hopkins University School of Medicine)
+ 2 AuthorsLinzhao Cheng46
Estimated H-index: 46
Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated β-globin alleles (βs/βs). Using a gene-targeting plasmid containing a loxP-flanked drug-resi...
226 Citations Source Cite
Published on Mar 1, 2008in Molecular Therapy 7.01
Nancy M. P. King21
Estimated H-index: 21
(Wake Forest University),
Odile Cohen-Haguenauer6
Estimated H-index: 6
(École normale supérieure de Cachan)
In Geneva, Switzerland, on 2–3 April 2007, Clinigene-NoE (the European Network for the Advancement of Clinical Gene Transfer and Therapy) and Consert (program on Concerted Safety and Efficiency Evaluation of Retroviral Transgenesis for Gene Therapy of Inherited Diseases)—two European Union (EU) programs that aim to facilitate the development of sound, safe, and effective human gene transfer—held a joint “think tank” on the ethics of human clinical gene transfer (GT).1
21 Citations Source Cite
Published on May 1, 2002in Nature Biotechnology 35.72
Makoto Miyagishi47
Estimated H-index: 47
(University of Tokyo),
Kazunari Taira58
Estimated H-index: 58
(National Institute of Advanced Industrial Science and Technology)
U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells
749 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 Jun 1, 2005in Nature 41.58
Fyodor D. Urnov42
Estimated H-index: 42
(Sangamo BioSciences),
Jeffrey C. Miller35
Estimated H-index: 35
(Sangamo BioSciences)
+ 7 AuthorsMichael C. Holmes45
Estimated H-index: 45
(Sangamo BioSciences)
Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a...
1,270 Citations Source Cite
  • References (46)
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Cited By4240
Published on Dec 1, 2013in Biochemical Society Transactions 3.39
Tautvydas Karvelis7
Estimated H-index: 7
(Vilnius University),
Giedrius Gasiunas13
Estimated H-index: 13
(Vilnius University),
Virginijus Siksnys32
Estimated H-index: 32
(Vilnius University)
The ternary Cas9–crRNA–tracrRNA complex (Cas9t) of the Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) system functions as an Mg 2+ -dependent RNA-directed DNA endonuclease that locates its DNA target guided by the crRNA (CRISPR RNA) in the tracrRNA–crRNA structure and introduces a double-strand break at a specific site in DNA. The simple modular organization of Cas9t, where specificity for the DNA target is encoded by a small crRNA and the clea...
14 Citations Source Cite
Published on Jan 1, 2014in Methods in Enzymology 1.98
Lianne E.M. Vriend6
Estimated H-index: 6
(University of Amsterdam),
Maria Jasin77
Estimated H-index: 77
(Memorial Sloan Kettering Cancer Center),
Przemek M. Krawczyk15
Estimated H-index: 15
(University of Amsterdam)
Thousands of DNA breaks occur daily in mammalian cells, including potentially tumorigenic double-strand breaks (DSBs) and less dangerous but vastly more abundant single-strand breaks (SSBs). The majority of SSBs are quickly repaired, but some can be converted to DSBs, posing a threat to the integrity of the genome. Although SSBs are usually repaired by dedicated pathways, they can also trigger homologous recombination (HR), an error-free pathway generally associated with DSB repair. While HR-med...
9 Citations Source Cite
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 Jan 1, 2014in Advances in Botanical Research 1.39
Robert J. Schmitz37
Estimated H-index: 37
(University of Georgia),
Xiaoyu Zhang28
Estimated H-index: 28
(University of Georgia)
Abstract One of the most exciting findings in recent years is the discovery that the two main components of the chromatin, DNA and histones, can both be covalently modified by a myriad of pathways. The combination of high-throughput technologies and well-established biochemical assays has made it possible to analyse chromatin modifications at a genome-wide level with increasingly high resolution. As described in this chapter, results from these ‘epigenomics’ studies have significantly broadened ...
2 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 Jan 1, 2015in Methods of Molecular Biology
Makiko Meguro-Horike6
Estimated H-index: 6
(Kanazawa University),
Shin-ichi Horike13
Estimated H-index: 13
(Kanazawa University)
Recent evidence implicated several long noncoding RNA (lncRNA) in gene expression in cis or trans through regulating the local chromosomal architecture. However, the mechanisms underlying the lncRNA mediated silencing of multiple genes remain unknown. We believe that Microcell Mediated Chromosome Transfer (MMCT) is a suitable approach for functional analysis of lncRNAs and nuclear dynamics. MMCT is a unique research technique that can be generally used to transfer a single chromosome from one ma...
1 Citations Source Cite
Published on Jan 1, 2014in Methods of Molecular Biology
Marlene Belfort53
Estimated H-index: 53
(University at Albany, SUNY),
Richard P. Bonocora1
Estimated H-index: 1
(Wadsworth Center)
Homing endonucleases are strong drivers of genetic exchange and horizontal transfer of both their own genes and their local genetic environment. The mechanisms that govern the function and evolution of these genetic oddities have been well documented over the past few decades at the genetic, biochemical, and structural levels. This wealth of information has led to the manipulation and reprogramming of the endonucleases and to their exploitation in genome editing for use as therapeutic agents, fo...
29 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 Jan 1, 2014in Methods of Molecular Biology
Neena Pyzocha5
Estimated H-index: 5
(Massachusetts Institute of Technology),
F. Ann Ran10
Estimated H-index: 10
(Massachusetts Institute of Technology)
+ 1 AuthorsFeng Zhang101
Estimated H-index: 101
(Massachusetts Institute of Technology)
The microbial CRISPR-Cas adaptive immune system can be harnessed to facilitate genome editing in eukaryotic cells (Cong L et al., Science 339, 819–823, 2013; Mali P et al., Science 339, 823–826, 2013). Here we describe a protocol for the use of the RNA-guided Cas9 nuclease from the Streptococcus pyogenes type II CRISPR system to achieve specifi c, scalable, and cost-effi cient genome editing in mammalian cells.
22 Citations Source Cite