Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

被引：812

作者：

Hendel, Ayal ^{[1
]}

Bak, Rasmus O. ^{[1
]}

Clark, Joseph T. ^{[1
]}

Kennedy, Andrew B. ^{[2
]}

Ryan, Daniel E. ^{[2
]}

Roy, Subhadeep ^{[3
]}

Steinfeld, Israel ^{[4
]}

Lunstad, Benjamin D. ^{[3
]}

Kaiser, Robert J. ^{[2
]}

Wilkens, Alec B. ^{[1
]}

Bacchetta, Rosa ^{[1
]}

Tsalenko, Anya ^{[2
]}

Dellinger, Douglas ^{[3
]}

Bruhn, Laurakay ^{[2
]}

Porteus, Matthew H. ^{[1
]}

机构：

[1] Stanford Univ, Dept Pediat, Stanford, CA 94305 USA

[2] Agilent Res Labs, Santa Clara, CA USA

[3] Agilent Res Labs, Boulder, CO USA

[4] Agilent Res Labs, Tel Aviv, Israel

来源：

NATURE BIOTECHNOLOGY | 2015年 / 33卷 / 09期

基金：

美国国家卫生研究院;

关键词：

SOLID-PHASE; T-CELLS; CCR5; OLIGONUCLEOTIDES; PHOSPHONOACETATE; SYSTEMS; GLOBIN; GENES; LOCI; HIV;

D O I：

10.1038/nbt.3290

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

引用

收藏

页码：985 / U232

页数：7

相关论文

共 24 条

[1] Easy quantitative assessment of genome editing by sequence trace decomposition [J].

Brinkman, Eva K. ;

Chen, Tao ;

Amendola, Mario ;

van Steensel, Bas .

NUCLEIC ACIDS RESEARCH, 2014, 42 (22)

[2] Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System [J].

Chen, Baohui ;

Gilbert, Luke A. ;

Cimini, Beth A. ;

Schnitzbauer, Joerg ;

Zhang, Wei ;

Li, Gene-Wei ;

Park, Jason ;

Blackburn, Elizabeth H. ;

Weissman, Jonathan S. ;

Qi, Lei S. ;

Huang, Bo .

CELL, 2013, 155 (07) :1479-1491

[3] Multiplex Genome Engineering Using CRISPR/Cas Systems [J].

Cong, Le ;

Ran, F. Ann ;

Cox, David ;

Lin, Shuailiang ;

Barretto, Robert ;

Habib, Naomi ;

Hsu, Patrick D. ;

Wu, Xuebing ;

Jiang, Wenyan ;

Marraffini, Luciano A. ;

Zhang, Feng .

SCIENCE, 2013, 339 (6121) :819-823

[4] COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites [J].

Cradick, Thomas J. ;

Qiu, Peng ;

Lee, Ciaran M. ;

Fine, Eli J. ;

Bao, Gang .

MOLECULAR THERAPY-NUCLEIC ACIDS, 2014, 3 :e214

[5] CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity [J].

Cradick, Thomas J. ;

Fine, Eli J. ;

Antico, Christopher J. ;

Bao, Gang .

NUCLEIC ACIDS RESEARCH, 2013, 41 (20) :9584-9592

[6] Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing [J].

Deleavey, Glen F. ;

Damha, Masad J. .

CHEMISTRY & BIOLOGY, 2012, 19 (08) :937-954

[7] Solid-phase chemical synthesis of phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides [J].

Dellinger, DJ ;

Sheehan, DM ;

Christensen, NK ;

Lindberg, JG ;

Caruthers, MH .

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2003, 125 (04) :940-950

[8] Streamlined Process for the Chemical Synthesis of RNA Using 2′-O-Thionocarbamate-Protected Nucleoside Phosphoramidites in the Solid Phase [J].

Dellinger, Douglas J. ;

Timar, Zoltan ;

Myerson, Joel ;

Sierzchala, Agnieszka B. ;

Turner, John ;

Ferreira, Fernando ;

Kupihar, Zoltan ;

Dellinger, Geraldine ;

Hill, Kenneth W. ;

Powell, James A. ;

Sampson, Jeffrey R. ;

Caruthers, Marvin H. .

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2011, 133 (30) :11540-11556

[9] Phosphorothioates, Essential Components of Therapeutic Oligonucleotides [J].

Eckstein, Fritz .

NUCLEIC ACID THERAPEUTICS, 2014, 24 (06) :374-387

[10] Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation [J].

Gilbert, Luke A. ;

Horlbeck, Max A. ;

Adamson, Britt ;

Villalta, Jacqueline E. ;

Chen, Yuwen ;

Whitehead, Evan H. ;

Guimaraes, Carla ;

Panning, Barbara ;

Ploegh, Hidde L. ;

Bassik, Michael C. ;

Qi, Lei S. ;

Kampmann, Martin ;

Weissman, Jonathan S. .

CELL, 2014, 159 (03) :647-661