Objective: To construct a W203X-mutant mouse model of cblC type methylmalonic acidemia based on the CRISPR/Cas9 technology.
Methods: At first, BLAST was used to compare the conservative nature of the cblC gene and protein sequences in humans and mice, and then, the CRISPR/Cas9 technology was used for microinjection of mouse fertilized eggs to obtain heterozygous F1 mice. Hybridization was performed for these mice to obtain homozygous W203X-mutant mice. The blood level of the metabolite propionyl carnitine (C3) was measured for homozygous mutant mice, heterozygous littermates, and wild-type mice.
Results: The gene and protein sequences of MMACHC, the pathogenic gene for cblC type methylmalonic acidemia, were highly conserved in humans and mice. The homozygous W203X-mutant mice were successfully obtained by the CRISPR/Cas9 technology, and there was a significant increase in C3 in these mice at 24 hours after birth (P<0.001).
Conclusions: A W203X-mutant mouse model of cblC type methylmalonic acidemia is successfully constructed by the CRISPR/Cas9 technology.
目的: 利用CRISPR/Cas9技术构建与人甲基丙二酸血症cblC型W203X突变型一致的小鼠模型。
方法: 通过BLAST比对人和小鼠cblC基因和蛋白序列的保守性，应用CRISPR/Cas9技术进行小鼠受精卵显微注射，获得杂合子F1代小鼠，F1代小鼠通过杂交获得W203X纯合突变型小鼠，并对纯合突变型、同窝杂合型及野生型3种类型小鼠进行血代谢产物丙酰肉碱检测。
结果: 人和小鼠甲基丙二酸血症cblC型的致病基因MMACHC的核苷酸和氨基酸序列高度保守。通过CRISPR/Cas9技术成功获得W203X纯合突变型小鼠，该小鼠模型在生后24 h丙酰肉碱明显升高（P < 0.001）。
结论: 利用CRISPR/Cas9技术成功构建了与人甲基丙二酸血症cblC型W203X突变型一致的小鼠模型。