优化sgRNA序列提高斑马鱼CRISPR/Cas9系统的突变效率

Enhancing the Mutative Efficiency of CRISPR/Cas9 System by Optimizing the sgRNA Sequences in Zebrafish

CRISPR/Cas9系统介导的定点突变已经在多种模式生物中实现,其中提高sgRNA的编辑效率是关键性因素。通过分析斑马鱼Danio rerio sgRNA序列特征,优化现有sgRNA设计原则,提高了CRISPR/Cas9系统在斑马鱼中的切割效率。根据现有的sgRNA设计原则,利用软件CHOPCHOP和Benchling对斑马鱼内35个基因设计157个sgRNAs并分析其序列特征对基因突变效率的影响。结果表明,CRISPR/Cas9系统对斑马鱼产生高效靶向突变的sgRNA序列具有明显的碱基偏好性。当靶点序列和靶点种子序列的GC含量为50%~60%、紧邻靶点3’端的前间区序列邻近基序可变碱基为C、Cas9蛋白复合体的切割位点碱基对为AC时,靶点序列具有较高的切割效率。同时,对6个已发表数据集的959个sgRNAs的序列特征进行重新分析,得到了类似的结果。优化的sgRNA设计原则,能显著提高靶基因的突变效率。

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis.The key point of this technology is to improve the gene editing efficiency of sgRNA.Optimizing the existing design principles by analyzing sgRNA sequence characteristics in zebrafish has enhanced the cleavage efficiency of CRISPR/Cas9 system.In this study,CHOPCHOP and Benchling software were used to design 157 sgRNAs for 35 genes in accordance with the existing sgRNA design principles,and then the influence of sequence features on gene mutative efficiency were analyzed.The results showed that CRISPR/Cas9 system showed obvious base preference for the sgRNA sequences that produced highly targeted mutations in zebrafish.Specifically,the target sequences have a higher mutative efficiency when the GC content in both target and seed sequences was 50%-60%,the protospacer adjacent motif variable base was cytosine and the target cleavage site base pair of the Cas9 protein complex was AC.Moreover,the sequence characteristics of 959 sgRNAs in 6 published data sets were reanalyzed and the similar results were obtained.The optimized sgRNA design principle can significantly increase the mutative efficiency of target genes.