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RNA-Guided Human Genome Engineering via Cas9

Published on Feb 15, 2013in Science 41.06
· DOI :10.1126/science.1232033
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 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 (4460)
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 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.
1,414 Citations Source Cite
Published on Aug 17, 2012in Science 41.06
Martin Jinek26
Estimated H-index: 26
(University of California, Berkeley),
Krzysztof Chylinski6
Estimated H-index: 6
(Umeå University)
+ 3 AuthorsEmmanuelle Charpentier28
Estimated H-index: 28
(Umeå University)
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in ...
4,942 Citations Source Cite
Published on Jun 13, 2012in PLOS Genetics 5.54
Mina Rho16
Estimated H-index: 16
(Indiana University Bloomington),
Yu Wei Wu12
Estimated H-index: 12
(Indiana University Bloomington)
+ 2 AuthorsYuzhen Ye29
Estimated H-index: 29
(Indiana University Bloomington)
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with cas (CRISPR–associated) genes, form the CRISPR/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human M...
88 Citations Source Cite
Rodolphe Barrangou50
Estimated H-index: 50
Philippe Horvath27
Estimated H-index: 27
Bacteria have been widely used as starter cultures in the food industry, notably for the fermentation of milk into dairy products such as cheese and yogurt. Lactic acid bacteria used in food manufacturing, such as lactobacilli, lactococci, streptococci, Leuconostoc, pediococci, and bifidobacteria, are selectively formulated based on functional characteristics that provide idiosyncratic flavor and texture attributes, as well as their ability to withstand processing and manufacturing conditions. U...
123 Citations Source Cite
Published on Feb 1, 2012in Nature 41.58
Blake Wiedenheft29
Estimated H-index: 29
(Howard Hughes Medical Institute),
Samuel H. Sternberg20
Estimated H-index: 20
(University of California, Berkeley),
Jennifer A. Doudna92
Estimated H-index: 92
(Lawrence Berkeley National Laboratory)
993 Citations Source Cite
Published on Jan 1, 2012in Nature Protocols 12.42
Neville E. Sanjana21
Estimated H-index: 21
(McGovern Institute for Brain Research),
Le Cong15
Estimated H-index: 15
(McGovern Institute for Brain Research)
+ 3 AuthorsFeng Zhang104
Estimated H-index: 104
(McGovern Institute for Brain Research)
458 Citations Source Cite
Published on Jan 1, 2012in Nucleic Acids Research 11.56
Timothy R. Dreszer14
Estimated H-index: 14
(University of California, Santa Cruz),
Donna Karolchik35
Estimated H-index: 35
(University of California, Santa Cruz)
+ 26 AuthorsKate R. Rosenbloom25
Estimated H-index: 25
(University of California, Santa Cruz)
The University of California Santa Cruz Genome Browser ( offers online public access to a growing database of genomic sequence and annotations for a wide variety of organisms. The Browser is an integrated tool set for visualizing, comparing, analyzing and sharing both publicly available and user-generated genomic data sets. In the past year, the local database has been updated with four new species assemblies, and we anticipate another four will be released by the end of 2...
274 Citations Source Cite
Published on Dec 15, 2011in Annual Review of Genetics 9.59
Devaki Bhaya30
Estimated H-index: 30
(Stanford University),
Michelle Davison6
Estimated H-index: 6
(Stanford University),
Rodolphe Barrangou50
Estimated H-index: 50
Bacteria and archaea have evolved defense and regulatory mechanisms to cope with various environmental stressors, including virus attack. This arsenal has been expanded by the recent discovery of the versatile CRISPR-Cas system, which has two novel features. First, the host can specifically incorporate short sequences from invading genetic elements (virus or plasmid) into a region of its genome that is distinguished by clustered regularly interspaced short palindromic repeats (CRISPRs). Second, ...
479 Citations Source Cite
Published on Dec 1, 2011in Algorithms for Molecular Biology 1.54
Ronny Lorenz16
Estimated H-index: 16
(University of Vienna),
Stephan H. Bernhart25
Estimated H-index: 25
(University of Vienna)
+ 4 AuthorsIvo L. Hofacker58
Estimated H-index: 58
(University of Copenhagen)
Background Secondary structure forms an important intermediate level of description of nucleic acids that encapsulates the dominating part of the folding energy, is often well conserved in evolution, and is routinely used as a basis to explain experimental findings. Based on carefully measured thermodynamic parameters, exact dynamic programming algorithms can be used to compute ground states, base pairing probabilities, as well as thermodynamic properties.
1,222 Citations Source Cite
Cited By4460
Published on Feb 28, 2019in Genome Medicine 8.90
Rabia Begum1
Estimated H-index: 1
Source Cite
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 Feb 12, 2019in Scientific Reports 4.12
Luis G. V. Fernandes7
Estimated H-index: 7
(Instituto Butantan),
L. P. Guaman + 3 AuthorsAna L. T. O. Nascimento29
Estimated H-index: 29
(Instituto Butantan)
Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the de...
Source Cite
Published on Mar 8, 2019in Scientific Reports 4.12
Ajeet Kaushik35
Estimated H-index: 35
(Florida International University),
Adriana Yndart11
Estimated H-index: 11
(Florida International University)
+ 7 AuthorsMadhavan Nair39
Estimated H-index: 39
(Florida International University)
CRISPR-Cas9/gRNA exhibits therapeutic efficacy against latent human immunodeficiency virus (HIV) genome but the delivery of this therapeutic cargo to the brain remains as a challenge. In this research, for the first time, we demonstrated magnetically guided non-invasive delivery of a nano-formulation (NF), composed of Cas9/gRNA bound with magneto-electric nanoparticles (MENPs), across the blood-brain barrier (BBB) to inhibit latent HIV-1 infection in microglial (hμglia)/HIV (HC69) cells. An opti...
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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 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