高效切割Hipp11敲入位点的sgRNA设计及活性验证

Designation and Activity Verification of High Efficiency sgRNAs for Hipp11 Locus

目的设计并验证能够高效切割Hipp11位点的sgRNA,为在Hipp11位点定点敲入外源基因提供工作基础。方法使用预测软件对Hipp11位点的sgRNA进行预测并挑选脱靶效应较低的两个sgRNA。构建相应载体,通过CRISPR/Cas9活性检测试剂盒在体外检测sgRNA的活性。选取活性较高的sgRNA体外转录,与Cas9 m RNA一并注射受精卵。检测出生后小鼠Hipp11位点切割效率,计算出sgRNA的体内活性。结果两个sgRNA的体外活性分别为19.2%和51.7%,使用体外活性高的2号sgRNA显微注射获得8只新生小鼠,其中62.5%(5/8)的小鼠中检测到Hipp11位点周围发生碱基插入或缺失。结论获得一个能够引导高效切割的Hipp11位点通用型sgRNA,从而为基因CRISPR技术在Hipp11位点定点插入外源基因奠定基础。sgRNA体外活性能够预测sgRNA在体内的切割活性,表明体外活性验证是筛选高活性sgRNA的有效手段。

Objective Designed and verified sgRNAs for Hipp11 locus,providing working foundation for gene knock-in in Hipp11 locus. Method Predicted sgRNAs for Hipp11 locus by software. Two sgRNAs with lowest offtarget effect were selected for vector construction. CRISPR / Cas9 activity kit was used to test relative activity of sgRNAs in vitro. sgRNA with relative activity of 51. 7% was subjected to in vitro transcription and was microinjected with Cas9 m RNA into zygotes. The cleavage activity of new born mice were tested to obtain sgRNA activity in vivo. Result The activity of two sgRNAs was 19. 2% and 51. 7% respectively in vitro. 2# sgRNA with higher in vitro activity was used from microinjection. Eight mice were born,among which 62. 5%( 5 /8) of the cleavage sites had base pairs insertion / deletion. Conclusion A sgRNA with high activity targeted Hipp11 locus was obtained,providing working foundation for gene knock-in by CRISPR / Cas9 technique in this locus. The activity testing in vitro can efficiently predict the result in vivo. So verification activity of sgRNA in vitro was an efficient method for sgRNA screening.