CAJ | 학술논문

CRISPR/Cas9技术是新近发展起来的对细胞和动物模型进行基因编辑的重要方法。本文利用DNA双链断裂(Double-strand breaks, DSBs)引起的同源重组(Homologous recombination, HR)依赖与非依赖的修复机制，建立基于 CRISPR/Cas9核酸酶技术构建定点突变小鼠品系的技术体系。针对赖氨酸特异脱甲基化酶2b(Lysine (K)-specific demethylase 2b, Kdm2b)酶活关键位点对应的基因组DNA序列设计单一导向RNA(Single-guide RNA, sgRNA)，通过与Cas9 mRNA共显微注射，分别得到Kdm2b基因发生移码突变的基因失活品系及关键位点氨基酸缺失的酶活突变型小鼠品系。此外，利用 HR 介导的修复机理，将黄素单加氧酶3(Flavin containing mo-nooxygenases3, Fmo3)基因的sgRNA序列及对应的点突变单链寡脱氧核苷(Single strand oligonucleotides, ssODN)修复模板共注射到小鼠受精卵雄原核。对F0小鼠基因测序分析显示，成功构建了Fmo3基因移码突变的基因敲除和单碱基定点突变的基因敲入小鼠，这些突变能够稳定遗传给子代。本研究利用CRISPR/Cas9技术，通过同源重组依赖与非依赖两种DNA损伤修复方式，成功构建了特定位点突变的小鼠品系。
CRISPR/Cas9기술시신근발전기래적대세포화동물모형진행기인편집적중요방법。본문이용DNA쌍련단렬(Double-strand breaks, DSBs)인기적동원중조(Homologous recombination, HR)의뢰여비의뢰적수복궤제，건립기우 CRISPR/Cas9핵산매기술구건정점돌변소서품계적기술체계。침대뢰안산특이탈갑기화매2b(Lysine (K)-specific demethylase 2b, Kdm2b)매활관건위점대응적기인조DNA서렬설계단일도향RNA(Single-guide RNA, sgRNA)，통과여Cas9 mRNA공현미주사，분별득도Kdm2b기인발생이마돌변적기인실활품계급관건위점안기산결실적매활돌변형소서품계。차외，이용 HR 개도적수복궤리，장황소단가양매3(Flavin containing mo-nooxygenases3, Fmo3)기인적sgRNA서렬급대응적점돌변단련과탈양핵감(Single strand oligonucleotides, ssODN)수복모판공주사도소서수정란웅원핵。대F0소서기인측서분석현시，성공구건료Fmo3기인이마돌변적기인고제화단감기정점돌변적기인고입소서，저사돌변능구은정유전급자대。본연구이용CRISPR/Cas9기술，통과동원중조의뢰여비의뢰량충DNA손상수복방식，성공구건료특정위점돌변적소서품계。
The CRISPR/Cas9 system is a recently developed important technology for genome editing in cellular and animal models. Here we established a CRISPR/Cas9-based system of generating site-specific mutant mice using DNA double-strand breaks (DSBs) induced homologous recombination (HR)-dependent or independent repair me-chanism. Through co-microinjection of Cas9 mRNA and single-guide RNA (sgRNA) targeting genomic DNA se-quence corresponding to enzyme activity of lysine (K)-specific demethylase 2b (Kdm2b), both a frame-shifted Kdm2b null mutant and a Kdm2b enzyme activity disrupted mouse strain were obtained simultaneously. Moreover, sgRNA targeting flavin containing monooxygenases3 (Fmo3) gene and the corresponding single strand oligonucleotides (ssODN) donor template with point mutation were co-injected into the male pronucleus of one-cell mouse embryos stimulated HR-mediated repair mechanism. Genomic sequence analysis of F0 mice showed that frame-shifted Fmo3 knockout mouse and site-specific Fmo3 knock-in mouse with single base substitution were successfully generated, and these mutations could be stably transmitted to the next generation. Therefore, we successfully generated mouse strains containing site-specific mutations through HR-dependent and -independent DSB repair using the CRISPR/Cas9 system.