Genome editing with FnCas12a using crRNA with altered target length in Oryza sativa

The crRNA of FnCas12a generally has a 24-nt guide sequence that is complementary to the target DNA sequence. Previous reports and our studies have shown that changing the length of guide sequence affects genome editing efficiency in rice. The goal of this project is to reveal the effects of guide sequence length on mutation pattern and efficiency. We constructed crRNAs with 18-nt, 24-nt, and 30-nt guide sequence and assessed the deletion mutation pattern and mutation efficiency at five target sites in rice genes by amplicon deep sequencing analysis. This study will facilitate establishing a high efficiently and precise CRISPR/FnCas12a-mediated genome editing system.

FnCas12a的crRNA（CRISPR RNA）通常带有一段与靶DNA序列互补的24 nt向导序列（guide sequence）。已有研究报道及本团队的研究均证实，改变向导序列的长度会影响水稻中的基因组编辑效率。本项目旨在阐明向导序列长度对突变模式及编辑效率的影响。我们构建了带有18 nt、24 nt及30 nt向导序列的crRNA，并通过扩增子深度测序（amplicon deep sequencing）分析，对水稻基因内5个靶位点的缺失突变模式及突变效率进行了评估。本研究将有助于构建高效且精准的CRISPR/FnCas12a介导的基因组编辑体系。