Cheng L H, Liu Y, Niu T
Department of Hematology and Research Laboratory of Hematology, West China Hospital, Sichuan University, Chengdu 610041, China.
Zhonghua Xue Ye Xue Za Zhi. 2017 May 14;38(5):427-431. doi: 10.3760/cma.j.issn.0253-2727.2017.05.014.
Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. 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. 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. Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.
利用CRISPR-Cas9基因编辑技术在同一条染色体上实现多个基因的共缺失。通过分子克隆构建了可诱导小鼠11B3染色体上Aloxe3-Alox12b-Alox8基因簇缺失的CRISPR-Cas9慢病毒质粒。转染HEK293T细胞以包装CRISPR或Cas9 cDNA的慢病毒，然后用慢病毒感染小鼠NIH3T3细胞并提取这些细胞的基因组DNA。通过PCR扩增缺失片段，利用TA克隆、桑格测序等技术确认Aloxe3-Alox12b-Alox8基因簇的缺失。成功构建了可诱导Aloxe3-Alox12b-Alox8基因簇缺失的CRISPR-Cas9慢病毒质粒。通过PCR验证了目标染色体片段（Aloxe3-Alox12b-Alox8基因簇）的缺失。通过TA克隆、桑格测序确认了Aloxe3-Alox12b-Alox8基因簇的缺失，CRISPR-Cas9系统介导切割事件的断点连接精确重组，两个切割位点之间完全未发生插入突变。CRISPR-Cas9基因编辑系统成功诱导了小鼠11B3染色体上Aloxe3-Alox12b-Alox8基因簇的大片段缺失。
