State Key Laboratory of Plant Cell and Chromosome Engineering, and Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
University of Chinese Academy of Sciences, Beijing, China.
Genome Biol. 2017 Oct 11;18(1):191. doi: 10.1186/s13059-017-1325-9.
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.
高保真 SpCas9 变体（eSpCas9 和 SpCas9-HF1）经过工程改造以降低脱靶效应。我们发现，向导 RNA 长度的变化导致植物细胞中 SpCas9 变体的编辑活性显著降低。精确匹配 20 个核苷酸的向导序列的单向导 RNA 对于 eSpCas9 和 SpCas9-HF1 的高靶编辑活性是必需的。来自 tRNA-sgRNA 前体的精确 20 个核苷酸向导序列使这些变体具有增强的特异性，从而能够进行强大的靶编辑。我们的工作揭示了一种有效增强高保真 SpCas9 核酸酶用于高效和精确基因组工程的方法。
