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RNA-Guided Human Genome Engineering via Cas9

Science 15 February 2013: 
Vol. 339 no. 6121 pp. 823-826 
DOI: 10.1126/science.1232033


Prashant Mali1,*, Luhan Yang1,3,*, Kevin M. Esvelt2, John Aach1, Marc Guell1, James E. DiCarlo4,Julie E. Norville1, George M. Church1,2,†1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.2Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138, USA.3Biological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02115, USA.4Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.↵†To whom correspondence should be addressed. E-mail: gchurch{at}genetics.med.harvard.edu

↵* These authors contributed equally to this work.


Karen Lin's insight:

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.

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One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering

One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering | KLin's reading | Scoop.it

Wang et al., 2013, Cell

 

Highlights

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> CRISPR/Cas9-mediated simultaneous targeting of five genes in mES cells

> Generation of Tet1/Tet2 double-mutant mice in one step

> Generation of Tet1/Tet2 double-mutant mice with predefined mutations in one step

 

Summary

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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) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.


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wb's curator insight, May 6, 2013 10:31 AM

amazing.....

Rescooped by Karen Lin from CRISPR-Cas System for Eukaryotic Genome Engineering
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Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems

Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems | KLin's reading | Scoop.it

Li et al., 2013, Nat Biotech

 

CRISPRs are clustered, regularly interspaced, short palindromic repeats present in many bacteria and archaea genomes. Proteins encoded by CRISPR-associated (Cas) genes serve as guardians of the genome, which target foreign DNA at specific sites by means of small CRISPR RNA (crRNA)-guided DNA recognition and degradation


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George Church - Keynote: Improving the Accuracy of Genome Sequencing and Interpretation

Watch on LabRoots at: http://labroots.com/user/webinars/details/id/29 Our ability to view and alter biology is progressing at an exponential pace -- faster e...
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A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity

A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity | KLin's reading | Scoop.it

Bacteriophages (or phages) are the most abundant biological entities on earth, and are estimated to outnumber their bacterial prey by tenfold. The constant threat of phage predation has led to the evolution of a broad range of bacterial immunity mechanisms that in turn result in the evolution of diverse phage immune evasion strategies, leading to a dynamic co-evolutionary arms race. Although bacterial innate immune mechanisms against phage abound, the only documented bacterial adaptive immune system is the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system, which provides sequence-specific protection from invading nucleic acids, including phage. Here we show a remarkable turn of events, in which a phage-encoded CRISPR/Cas system is used to counteract a phage inhibitory chromosomal island of the bacterial host. A successful lytic infection by the phage is dependent on sequence identity between CRISPR spacers and the target chromosomal island. In the absence of such targeting, the phage-encoded CRISPR/Cas system can acquire new spacers to evolve rapidly and ensure effective targeting of the chromosomal island to restore phage replication.


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High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells | KLin's reading | Scoop.it

Fu et al., 2013, Nat Biotech

Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell–based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.


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Freddy Monteiro's curator insight, June 24, 2013 9:26 PM

Well, at least it is not the end. The system can stll be improved. What about other organisms? Do they also present a similar rate of non-specific mutagenesis?

Freddy Monteiro's comment, June 24, 2013 9:41 PM
Clem Stanyon has a particularly relevant comparison with zinc-finger endonuculeases in his scoopit post: http://sco.lt/5BJsTh