限制性内切酶策略鉴定CRISPR/Cas9介导的Chrm3基因敲除小鼠

CRISPR/Cas9技术是近年发展起来的快速基因编辑技术。通过该技术已对多种生物的基因组进行了编辑。由此产生的基因编辑动物的建系与鉴定是随之而来较为繁琐的工作。单导向RNA(single-guide RNA, sgRNA)靶序列的设计和确定不仅影响后续靶向基因组的效率，还可作为优化鉴定、筛选方法的参考。本研究在选取sgRNA靶序列时，不仅依据软件的评分，还分析了sgRNA靶序列是否含有酶切位点，以便对后续纯合子/杂合子进行鉴定。结果显示，以特异引物扩增的野生型小鼠Chrm3基因片段可被限制性内切酶BanⅡ切为两个片段；而纯合子小鼠“丢失”该酶切位点，其PCR产物不能被切开；杂合子小鼠PCR产物被不完全切开，凝胶电泳结果可见三条带。本研究结果提示该策略可有效简化基因编辑动物建系鉴定工作，提高鉴定效率及改善阳性动物辨识效果。

Characterize CRISPR/Cas9 Established Chrm3 Gene Knockout Mice by Enzyme Restriction

CRISPR/Cas9-based gene editing technology has developed rapidly in recent years. Establishing lines of genomic modified animals requires the verification of founders. For mice generated using CRISPR/Cas9 system, this tedious procedure is also needed. By incorporating proper restriction sites in the design of single-guide RNA (sgRNA) can be helpful for the identification of establishment lines. To better verify homozygous and heterozygous mice, we selected sgRNA sequences by scores from predicting software and presence of specific restriction sites. We produced a genetically modified Chrm3 mice by cytoplasm micro injection. The PCR products from Chrm3 mice DNA were digested by BanⅡ. The results showed that the sample from WT mice had two bands after restriction|homozygous Chrm3 mice bearing no restriction sites showed only one band, and the heterozygotes gave three bands. Our attempted strategy, to some extent, simplified the line establishment verification and homozygous /heterozygote identification processes.