CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair

被引：153

作者：

Charpentier, M. ^{[1
]}

Khedher, A. H. Y. ^{[1
,2
]}

Menoret, S. ^{[3
]}

Brion, A. ^{[1
]}

Lamribet, K. ^{[1
]}

Dardillac, E. ^{[4
]}

Boix, C. ^{[1
]}

Perrouault, L. ^{[1
]}

Tesson, L. ^{[3
]}

Geny, S. ^{[1
]}

De Cian, A. ^{[1
]}

Itier, J. M. ^{[2
]}

Anegon, I. ^{[3
]}

Lopez, B. ^{[4
]}

Giovannangeli, C. ^{[1
]}

Concordet, J. P. ^{[1
]}

机构：

[1] Sorbonne Univ, CNRS UMR 7196, Museum Natl Hist Nat, INSERM U1154, 43 Rue Cuvier, F-75231 Paris, France

[2] Sanofi, Translat Sci, 13 Quai Jules Guesde, F-94400 Vitry Sur Seine, France

[3] Univ Nantes, CHU Nantes, INSERM, Ctr Rech Transplantat & Immunol UMR1064, 30 Ave Jean Monnet, F-44093 Nantes, France

[4] Univ Paris Saclay, Inst Cancerol Gustave Roussy, Equipe Labellisee Ligue Canc, CNRS UMR 8200, 114 Rue Edouard Vaillant, F-94805 Villejuif, France

来源：

NATURE COMMUNICATIONS | 2018年 / 9卷

关键词：

PLURIPOTENT STEM-CELLS; DNA-END RESECTION; ZINC-FINGER NUCLEASES; MAMMALIAN-CELLS; DIRECTED REPAIR; RECOMBINATION; GENOME; ENDONUCLEASE; REVEALS; DISRUPTION;

D O I：

10.1038/s41467-018-03475-7

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：

In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.

引用

页数：11

共 35 条

[1] Phosphorylated CtIP Functions as a Co-factor of the MRE11-RAD50-NBS1 Endonuclease in DNA End Resection [J].

Anand, Roopesh ;

Ranjha, Lepakshi ;

Cannavo, Elda ;

Cejka, Petr .

MOLECULAR CELL, 2016, 64 (05) :940-950

[2] Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases [J].

Beumer, Kelly J. ;

Trautman, Jonathan K. ;

Bozas, Ana ;

Liu, Ji-Long ;

Rutter, Jared ;

Gall, Joseph G. ;

Carroll, Dana .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2008, 105 (50) :19821-19826

[3] High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding [J].

Boyle, Evan A. ;

Andreasson, Johan O. L. ;

Chircus, Lauren M. ;

Sternberg, Samuel H. ;

Wu, Michelle J. ;

Guegler, Chantal K. ;

Doudna, Jennifer A. ;

Greenleaf, William J. .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2017, 114 (21) :5461-5466

[4] A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis [J].

Chenouard, Vanessa ;

Brusselle, Lucas ;

Heslan, Jean-Marie ;

Remy, Severine ;

Menoret, Severine ;

Usal, Claire ;

Ouisse, Laure-Helene ;

Tuan Huy NGuyen ;

Anegon, Ignacio ;

Tesson, Laurent .

JOURNAL OF GENETICS AND GENOMICS, 2016, 43 (05) :341-348

[5]

CHOULIKA A, 1995, MOL CELL BIOL, V15, P1968

[6] 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

[7] A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9 [J].

Dagdas, Yavuz S. ;

Chen, Janice S. ;

Sternberg, Samuel H. ;

Doudna, Jennifer A. ;

Yildiz, Ahmet .

SCIENCE ADVANCES, 2017, 3 (08)

[8] CtIP tetramer assembly is required for DNA-end resection and repair [J].

Davies, Owen R. ;

Forment, Josep V. ;

Sun, Meidai ;

Belotserkovskaya, Rimma ;

Coates, Julia ;

Galanty, Yaron ;

Demir, Mukerrem ;

Morton, Christopher R. ;

Rzechorzek, Neil J. ;

Jackson, Stephen P. ;

Pellegrini, Luca .

NATURE STRUCTURAL & MOLECULAR BIOLOGY, 2015, 22 (02) :150-157

[9] Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome [J].

DeKelver, Russell C. ;

Choi, Vivian M. ;

Moehle, Erica A. ;

Paschon, David E. ;

Hockemeyer, Dirk ;

Meijsing, Sebastiaan H. ;

Sancak, Yasemin ;

Cui, Xiaoxia ;

Steine, Eve Line J. ;

Miller, Jeffrey C. ;

Tam, Phillip ;

Bartsevich, Victor V. ;

Meng, Xiangdong ;

Rupniewski, Igor ;

Gopalan, Sunita M. ;

Sun, Helena C. ;

Pitz, Kathleen J. ;

Rock, Jeremy M. ;

Zhang, Lei ;

Davis, Gregory D. ;

Rebar, Edward J. ;

Cheeseman, Iain M. ;

Yamamoto, Keith R. ;

Sabatini, David M. ;

Jaenisch, Rudolf ;

Gregory, Philip D. ;

Urnov, Fyodor D. .

GENOME RESEARCH, 2010, 20 (08) :1133-1142

[10] Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases [J].

Doyon, Yannick ;

McCammon, Jasmine M. ;

Miller, Jeffrey C. ;

Faraji, Farhoud ;

Ngo, Catherine ;

Katibah, George E. ;

Amora, Rainier ;

Hocking, Toby D. ;

Zhang, Lei ;

Rebar, Edward J. ;

Gregory, Philip D. ;

Urnov, Fyodor D. ;

Amacher, Sharon L. .

NATURE BIOTECHNOLOGY, 2008, 26 (06) :702-708

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