In vivo genome editing using Staphylococcus aureus Cas9

被引：2040

作者：

Ran, F. Ann ^{[1
,2
]}

Cong, Le ^{[1
,3
]}

Yan, Winston X. ^{[1
,4
,5
]}

Scott, David A. ^{[1
,6
,7
]}

Gootenberg, Jonathan S. ^{[1
,8
]}

Kriz, Andrea J. ^{[3
]}

Zetsche, Bernd ^{[1
]}

Shalem, Ophir ^{[1
]}

Wu, Xuebing ^{[9
,10
]}

Makarova, Kira S. ^{[11
]}

Koonin, Eugene V. ^{[11
]}

Sharp, Phillip A. ^{[3
,9
]}

Zhang, Feng ^{[1
,6
,7
,12
]}

机构：

[1] Broad Inst MIT & Harvard, Cambridge, MA 02142 USA

[2] Harvard Univ, Soc Fellows, Cambridge, MA 02138 USA

[3] MIT, Dept Biol, Cambridge, MA 02139 USA

[4] Harvard Univ, Sch Med, Grad Program Biophys, Boston, MA 02115 USA

[5] Harvard Univ, Sch Med, Harvard MIT Div Hlth Sci & Technol, Boston, MA 02115 USA

[6] MIT, McGovern Inst Brain Res, Cambridge, MA 02139 USA

[7] MIT, Dept Brain & Cognit Sci, Cambridge, MA 02139 USA

[8] Harvard Univ, Sch Med, Dept Syst Biol, Boston, MA 02115 USA

[9] MIT, David H Koch Inst Integrat Canc Res, Cambridge, MA 02139 USA

[10] MIT, Computat & Syst Biol Grad Program, Cambridge, MA 02139 USA

[11] Natl Lib Med, Natl Ctr Biotechnol Informat, NIH, Bethesda, MD 20894 USA

[12] MIT, Dept Biol Engn, Cambridge, MA 02139 USA

来源：

NATURE | 2015年 / 520卷 / 7546期

基金：

美国国家卫生研究院; 美国国家科学基金会;

关键词：

OFF-TARGET SITES; GUIDE RNA; NUCLEASE SPECIFICITY; DNA CLEAVAGE; CRISPR RNA; GENE; IMMUNITY; SYSTEMS; ENDONUCLEASE; SEQUENCE;

D O I：

10.1038/nature14299

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

The RNA-guided endonuclease Cas9 has emerged as a versatile genom-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatileaderio-assolated virus (AAV) delivery vehicle. Here, we we characterize six smaller Cas9 ortholocgues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of spcas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection we observed >40%. gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol le; Is We further assess the genome -wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

引用

页码：186 / U98

页数：18

共 50 条

[41] In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9 [J].

Swiech, Lukasz ;

Heidenreich, Matthias ;

Banerjee, Abhishek ;

Habib, Naomi ;

Li, Yinqing ;

Trombetta, John ;

Sur, Mriganka ;

Zhang, Feng .

NATURE BIOTECHNOLOGY, 2015, 33 (01) :102-U286

[42] GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases [J].

Tsai, Shengdar Q. ;

Zheng, Zongli ;

Nguyen, Nhu T. ;

Liebers, Matthew ;

Topkar, Ved V. ;

Thapar, Vishal ;

Wyvekens, Nicolas ;

Khayter, Cyd ;

Iafrate, A. John ;

Le, Long P. ;

Aryee, Martin J. ;

Joung, J. Keith .

NATURE BIOTECHNOLOGY, 2015, 33 (02) :187-197

[43] Precise hit: Adeno-associated virus in gene targeting [J].

Vasileva, A ;

Jessberger, R .

NATURE REVIEWS MICROBIOLOGY, 2005, 3 (11) :837-847

[44]

Veldwijk MR, 2002, MOL THER, V6, P272, DOI 10.1006/mthe.2002.0569

[45] Mechanisms and optimization of in vivo delivery of lipophilic siRNAs [J].

Wolfrum, Christian ;

Shi, Shuanping ;

Jayaprakash, K. Narayanarmair ;

Jayaraman, Muthusamy ;

Wang, Gang ;

Pandey, Rajendra K. ;

Rajeev, Kallanthottathil G. ;

Nakayama, Tomoko ;

Charrise, Klaus ;

Ndungo, Esther M. ;

Zimmermann, Tracy ;

Koteliansky, Victor ;

Manoharan, Muthiah ;

Stoffel, Markus .

NATURE BIOTECHNOLOGY, 2007, 25 (10) :1149-1157

[46] Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells [J].

Wu, Xuebing ;

Scott, David A. ;

Kriz, Andrea J. ;

Chiu, Anthony C. ;

Hsu, Patrick D. ;

Dadon, Daniel B. ;

Cheng, Albert W. ;

Trevino, Alexandro E. ;

Konermann, Silvana ;

Chen, Sidi ;

Jaenisch, Rudolf ;

Zhang, Feng ;

Sharp, Phillip A. .

NATURE BIOTECHNOLOGY, 2014, 32 (07) :670-+

[47] RECENT PROGRESS IN UNDERSTANDING APOLIPOPROTEIN-B [J].

YOUNG, SG .

CIRCULATION, 1990, 82 (05) :1574-1594

[48] Model-based Analysis of ChIP-Seq (MACS) [J].

Zhang, Yong ;

Liu, Tao ;

Meyer, Clifford A. ;

Eeckhoute, Jerome ;

Johnson, David S. ;

Bernstein, Bradley E. ;

Nussbaum, Chad ;

Myers, Richard M. ;

Brown, Myles ;

Li, Wei ;

Liu, X. Shirley .

GENOME BIOLOGY, 2008, 9 (09)

[49] Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection [J].

Zincarelli, Carmela ;

Soltys, Stephen ;

Rengo, Giuseppe ;

Rabinowitz, Joseph E. .

MOLECULAR THERAPY, 2008, 16 (06) :1073-1080

[50] Mfold web server for nucleic acid folding and hybridization prediction [J].

Zuker, M .

NUCLEIC ACIDS RESEARCH, 2003, 31 (13) :3406-3415

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