利用CRISPR/Cas9技术体内标记示踪小鼠内源性NPC1L1蛋白

目的:对小鼠内源性NPC1L1蛋白进行标记和示踪，为研究体内胆固醇的吸收机制提供新的技术手段。方法:利用CRISPR/Cas9基因组编辑技术，针对小鼠NPC1L1基因的3′末端非编码区，设计不同sgRNA，利用细胞内的荧光素酶法，检测分析其Cas9/sgRNA核酸酶活性，筛选出高活性的sgRNA序列，经体外转录制备sgRNA和Cas9 mRNA。PCR扩增基因打靶的两侧同源臂，与FLAG-EGFP的编码cDNA一起克隆至打靶载体。经酶切和测序鉴定正确的打靶载体DNA与sgRNA、Cas9 mRNA一起显微注射入小鼠受精卵，移植至假孕小鼠体内自然发育；通过基因组DNA 的PCR和Southern 印迹分析，筛选、鉴定重组正确且无随机插入的子代小鼠。制备小肠组织冰冻切片，荧光显微镜下观察NPC1L1-EGFP的表达分布。结果:成功构建了NPC1L1基因敲入打靶载体，筛选了高活性的sgRNA，利用CRISPR/Cas9技术获得了对内源性NPC1L1蛋白C端进行FLAG和EGFP双重标记的子代小鼠，荧光显微镜下观察到该融合蛋白分布于小肠绒毛上皮细胞的刷状缘，与NPC1L1的分布特征一致。结论:成功实现了对小鼠内源性NPC1L1蛋白进行体内标记，为研究NPC1L1囊泡转运和胆固醇吸收机制提供了有效手段。

Genetic labeling and tracing of mouse endogenous NPC1L1 protein by CRISPR/Cas9 technology in vivo

Objective: To label and trace endogenous NPC1L1 protein in mouse model，providing a new tool for studying the absorption mechanisms of cholesterol in vivo. Methods:Using CRISPR/Cas9 genomic editing technology，different sgRNAs were designed according to the 3′ untranslated region of mouse NPC1L1 gene. The activity of Cas9/sgRNA nuclease was analyzed in cellular luciferase detection system，and the sgRNA sequence with high activity was screened out.The sgRNA and Cas9 mRNA were transcribed in vitro. The two homologous arms of the target gene were amplified by PCR and cloned into the target vector together with the cDNA encoding FLAG-EGFP. The correct targeting vector identified by restriction enzyme digestion and sequencing，along with sgRNA and cas9 mRNA were microinjected into fertilized eggs and then transplanted into pseudopregnant mice for natural development. The offspring mice with correct recombination and no random insertion were verified by PCR and Southern blotting analysis of genomic DNA. The expression of NPC1L1-FLAG-EGFP fusion protein was observed under fluorescence microscope using jejunal cryosections. Results: The FLAG-EGFP knock-in targeting vector of NPC1L1 gene was successfully constructed. The highly active sgRNA was determined，and the offspring mice with FLAG and EGFP dual-tags on the C-terminal of endogenous NPC1L1 protein were finally obtained by CRISPR/cas9 technology. The localization of the fusion protein along the brush border of villous epithelial cells in the small intestine was observed under fluorescence microscope，which was consistent with the distribution characteristics of NPC1L1. Conclusion: The successfully constructed mouse model with endogenous NPC1L1 protein labeling of dual-tags，FLAG and EGFP provides an effective tool to study the mechanism of vesicle transport and cholesterol absorption of NPC1L1 in vivo.