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

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-nu...

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Bibliographic Details
Published inGenome Biology Vol. 18; no. 1; p. 191
Main Authors Zhang, Dingbo, Zhang, Huawei, Li, Tingdong, Chen, Kunling, Qiu, Jin-Long, Gao, Caixia
Format Journal Article
LanguageEnglish
Published England BioMed Central 11.10.2017
BMC
Subjects
CRISPR
Deoxyribonucleic acid
DNA
Engineering
Fidelity
gene editing
Genome editing
Genomes
Method
Nuclease
nucleases
nucleotide sequences
Plant cells
Proteins
Ribonucleic acid
RNA
tRNA
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Summary: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.
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ISSN:1474-760X
1474-7596
1474-760X
DOI:10.1186/s13059-017-1325-9