Objective: Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. Methods: CRISPR-Cas9 lentiviral plasmid that could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse 11B3 chromosome was constructed via molecular clone. HEK293T cells were transfected to package lentivirus of CRISPR or Cas9 cDNA, then mouse NIH3T3 cells were infected by lentivirus and genomic DNA of these cells was extracted. The deleted fragment was amplified by PCR, TA clone, Sanger sequencing and other techniques were used to confirm the deletion of Aloxe3-Alox12b-Alox8 cluster genes. Results: The CRISPR-Cas9 lentiviral plasmid, which could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes, was successfully constructed. Deletion of target chromosome fragment (Aloxe3-Alox12b-Alox8 cluster genes) was verified by PCR. The deletion of Aloxe3-Alox12b-Alox8 cluster genes was affirmed by TA clone, Sanger sequencing, and the breakpoint junctions of the CRISPR-Cas9 system mediate cutting events were accurately recombined, insertion mutation did not occur between two cleavage sites at all. Conclusion: Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.
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。.
Keywords: CRISPR-Cas9; Chromosomal large deletion; Gene editing; Hematologic malignancies.