利用 CRISPR／Cas9技术构建 miRNA－29 b1基因敲除小鼠

摘要

Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank .sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C 57BL/6 mice.After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile , real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice .Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas 9.After microinjection, miRNA-29b1 gene-mutated mice were obtained.The sequencing results showed that there were two types of genotype for the mutated mice , one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion.Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly .Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR /Cas9 technology.%目的：应用CRISPR／Cas9技术构建miRNA－29b1基因敲除小鼠。方法针对miRNA－29b1基因设计一段sgRNA，sgRNA和Cas9体外转录后显微注射至C57BL／6小鼠受精卵细胞。小鼠出生后取其基因组DNA进行测序以鉴定基因型，同时取小鼠心、肝、脾、肺、肾等脏器研磨后提取总RNA，通过real－time PCR分析miRNA－29b1在这些脏器中的表达。结果设计了20 bp的miRNA－29b1sgRNA并与Cas9一起进行了体外转录，显微注射小鼠受精卵细胞后获得miRNA－29b1基因突变小鼠。测序结果表明突变小鼠有两种基因型，一种为10 bp的缺失突变；另一种为22 bp的缺失突变，同时伴有3 bp的插入突变。与野生型小鼠相比，基因突变小鼠心、肝、脾、肺、肾等组织中miRNA－29b1表达量下降明显。结论应用CRISPR／Cas9技术成功构建miRNA－29b1基因敲除小鼠。