Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases.
Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases.
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完美匹配的 20 核苷酸引导 RNA 序列可使用高保真 SpCas9 核酸酶进行稳健的基因组编辑。
DOI：
10.1186/s13059-017-1325-9
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发表时间：
2017-10-11
期刊：
影响因子：
12.3
通讯作者：
Gao C
中科院分区：
文献类型：
--
作者：
Zhang D;Zhang H;Li T;Chen K;Qiu JL;Gao C
高保真SpCas 9变体（eSpCas 9和SpCas 9-HF 1）已被工程化以减少脱靶效应。我们发现指导RNA长度的变化诱导植物细胞中SpCas 9变体的编辑活性显著降低。携带精确、完全匹配的20个核苷酸指导序列的单指导RNA对于eSpCas 9和SpCas 9-HF 1的高中靶编辑活性是必需的。源自tRNA-sgRNA前体的精确的20个核苷酸的指导序列能够通过这些变体进行稳健的靶向编辑，具有增强的特异性。我们的工作揭示了增强高保真SpCas 9核酸酶用于高效和精确基因组工程的有效方法。本文的在线版本（doi：10.1186/s13059-017-1325-9）包含补充材料，可供授权用户使用。
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. The online version of this article (doi:10.1186/s13059-017-1325-9) contains supplementary material, which is available to authorized users.