Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases

被引：104

作者：

Zhang, Dingbo ^{[1
,2
,3
]}

Zhang, Huawei ^{[1
,2
]}

Li, Tingdong ^{[1
,2
,3
]}

Chen, Kunling ^{[1
,2
]}

Qiu, Jin-Long ^{[4
]}

Gao, Caixia ^{[1
,2
]}

机构：

[1] Chinese Acad Sci, Inst Genet & Dev Biol, State Key Lab Plant Cell & Chromosome Engn, Beijing, Peoples R China

[2] Chinese Acad Sci, Inst Genet & Dev Biol, Ctr Genome Editing, Beijing, Peoples R China

[3] Univ Chinese Acad Sci, Beijing, Peoples R China

[4] Chinese Acad Sci, Inst Microbiol, State Key Lab Plant Genom, Beijing, Peoples R China

来源：

GENOME BIOLOGY | 2017年 / 18卷

基金：

中国国家自然科学基金;

关键词：

CRISPR-CAS NUCLEASES; HUMAN-CELLS; SPECIFICITY; SYSTEM; WHEAT; ENDONUCLEASES; VERSATILE; CLEAVAGE; PLANTS; RICE;

D O I：

10.1186/s13059-017-1325-9

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：

High-fidelity SpCas9 variants (eSpCas9 and SpCas9-HF1) have been engineered to reduce off-target effects. We found that changes in guide RNA length induced significant reductions in the editing activities of SpCas9 variants in plant cells. Single guide RNAs harboring precise, perfectly matched 20-nucleotide guide sequences are necessary for high on-target editing activities of eSpCas9 and SpCas9-HF1. Precise 20-nucleotide guide sequences derived from tRNA sgRNA precursors enable robust on-target editing by these variants with enhanced specificity. Our work reveals an effective way of enhancing the use of the high-fidelity SpCas9 nucleases for efficient and precise genome engineering.

引用

页数：7

共 42 条

[21] CRISPR-P: A Web Tool for Synthetic Single-Guide RNA Design of CRISPR-System in Plants [J].

Lei, Yang ;

Lu, Li ;

Liu, Hai-Yang ;

Li, Sen ;

Xing, Feng ;

Chen, Ling-Ling .

MOLECULAR PLANT, 2014, 7 (09) :1494-1496

[22] Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes [J].

Liang, Zhen ;

Chen, Kunling ;

Li, Tingdong ;

Zhang, Yi ;

Wang, Yanpeng ;

Zhao, Qian ;

Liu, Jinxing ;

Zhang, Huawei ;

Liu, Cuimin ;

Ran, Yidong ;

Gao, Caixia .

NATURE COMMUNICATIONS, 2017, 8

[23] CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering [J].

Mali, Prashant ;

Aach, John ;

Stranges, P. Benjamin ;

Esvelt, Kevin M. ;

Moosburner, Mark ;

Kosuri, Sriram ;

Yang, Luhan ;

Church, George M. .

NATURE BIOTECHNOLOGY, 2013, 31 (09) :833-+

[24] RNA-Guided Human Genome Engineering via Cas9 [J].

Mali, Prashant ;

Yang, Luhan ;

Esvelt, Kevin M. ;

Aach, John ;

Guell, Marc ;

DiCarlo, James E. ;

Norville, Julie E. ;

Church, George M. .

SCIENCE, 2013, 339 (6121) :823-826

[25] Guide RNA engineering for versatile Cas9 functionality [J].

Nowak, Chance M. ;

Lawson, Seth ;

Zerez, Megan ;

Bleris, Leonidas .

NUCLEIC ACIDS RESEARCH, 2016, 44 (20) :9555-9564

[26] High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity [J].

Pattanayak, Vikram ;

Lin, Steven ;

Guilinger, John P. ;

Ma, Enbo ;

Doudna, Jennifer A. ;

Liu, David R. .

NATURE BIOTECHNOLOGY, 2013, 31 (09) :839-+

[27] Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs [J].

Port, Fillip ;

Bullock, Simon L. .

NATURE METHODS, 2016, 13 (10) :852-+

[28] In vivo genome editing using Staphylococcus aureus Cas9 [J].

Ran, F. Ann ;

Cong, Le ;

Yan, Winston X. ;

Scott, David A. ;

Gootenberg, Jonathan S. ;

Kriz, Andrea J. ;

Zetsche, Bernd ;

Shalem, Ophir ;

Wu, Xuebing ;

Makarova, Kira S. ;

Koonin, Eugene V. ;

Sharp, Phillip A. ;

Zhang, Feng .

NATURE, 2015, 520 (7546) :186-U98

[29] Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity [J].

Ran, F. Ann ;

Hsu, Patrick D. ;

Lin, Chie-Yu ;

Gootenberg, Jonathan S. ;

Konermann, Silvana ;

Trevino, Alexandro E. ;

Scott, David A. ;

Inoue, Azusa ;

Matoba, Shogo ;

Zhang, Yi ;

Zhang, Feng .

CELL, 2013, 154 (06) :1380-1389

[30] Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery [J].

Rees, Holly A. ;

Komor, Alexis C. ;

Yeh, Wei-Hsi ;

Caetano-Lopes, Joana ;

Warman, Matthew ;

Edge, Albert S. B. ;

Liu, David R. .

NATURE COMMUNICATIONS, 2017, 8

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