Abstract
目的应用CRISPR-Cas9基因编辑技术来实现同一染色体片段上的多个基因共缺失。方法利用分子克隆技术构建能使小鼠染色体11B3上Aloxe3-Alox12b-Alox8基因簇缺失的CRISPR-Cas9慢病毒质粒；通过转染HEK293T细胞来包装带CRISPR或Cas9 cDNA的慢病毒，进而感染小鼠NIH3T3细胞，提取这些细胞的全基因组；通过PCR扩增缺失后的片段，TA克隆、Sanger测序等技术手段鉴定Aloxe3-Alox12b-Alox8基因簇缺失的情况。结果成功构建了能使Aloxe3-Alox12b-Alox8基因簇发生缺失的CRISPR-Cas9慢病毒质粒；PCR检测证实了目的染色体片段（Aloxe3-Alox12b-Alox8基因簇）的缺失；TA克隆和Sanger测序明确了Aloxe3-Alox12b-Alox8基因簇发生缺失，并且该CRISPR-Cas9系统介导的切割事件产生的断点被准确地连接在一起，在两个切割位点间没有发生插入突变。结论利用CRISPR-Cas9基因编辑技术可以有效实现小鼠染色体11B3上Aloxe3-Alox12b-Alox8基因簇的大片段缺失。
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