同源臂长度对CRISPR/Cas9介导hLF基因打靶山羊β-乳球蛋白位点效率的影响

Effects of homologous arm length on the efficiency of CRISPR/Cas9 mediated hLF gene knock-in at goatβ-lactoglobulin locus

【目的】探究同源臂长度对CRISPR/Cas9系统介导人乳铁蛋白基因(hLF)打靶山羊β-乳球蛋白基因(BLG)座位点效率的影响,为今后体细胞核移植制备BLG-/hLF+基因打靶山羊提供科学依据,也为CRISPR/Cas9基因编辑系统介导BLG基因或其他基因座位点定向精准分子修饰的遗传育种提供借鉴。【方法】针对山羊BLG基因第一外显子区域设计构建sgBLG/Cas9载体,电转染山羊胎儿成纤维细胞,PCR验证BLG基因座位点致突变活性;以BLC14乳腺特异性表达载体为基础构建3种同源臂长度(6.0、3.5和1.2 kb)的hLF基因打靶载体,分别与sgBLG/Cas9载体共转染山羊胎儿成纤维细胞,经500μg/mLG418筛选后,采用PCR检测基因打靶情况。【结果】sgBLG/Cas9载体在山羊胎儿成纤维细胞BLG基因座附近切割DNA双链的致突变活性效率在30%~35%。构建获得3种同源臂长度的hLF基因打靶载体(BLC14-1、BLC14-2和BLC14-3),对应的同源臂长度分别为6.0、3.5和1.2 kb;将3种hLF基因打靶载体与sgBLG/Cas9载体共转染山羊胎儿成纤维细胞,经5次电转染和G418筛选,分别获得83、77和86株药物抗性细胞,经PCR同源重组检测最终获得42、38和44株BLG-/hLF+基因打靶细胞株,即hLF基因在山羊胎儿成纤维细胞BLG基因座的平均打靶效率分别为50.6%(42/83)、49.4%(38/77)和51.2%(44/86)。3种不同长度同源臂构建的hLF基因打靶载体在山羊BLG基因座位点的打靶效率在统计学上无显著差异(P>0.05),表明同源臂长度对CRISPR/Cas9介导hLF基因打靶山羊BLG基因座位点无显著影响。【结论】利用CRISPR/Cas9系统介导hLF基因打靶山羊胎儿成纤维细胞BLG基因座位点能成功获得多株hLF+/BLG-基因打靶细胞株(BLG基因座定点打靶hLF基因),但打靶载体同源臂长度对CRISPR/Cas9系统介导BLG位点定向整合hLF基因的打靶效率无明显影响。

【Objective】To explore the effect of homology arm length on the efficiency of CRISPR/Cas9 system-mediated human lactoferrin gene(hLF)targeting goatβ-lactoglobulin gene(BLG)locus,and to provide a scientific basis for the preparation of BLG-/hLF+gene target cells by somatic cell nuclear transfer in the future.It also provided a reference for genetic breeding of BLG gene or other gene locus directed by precise molecular modification mediated by CRISPR/Cas9 gene editing system.【Method】According to the first exon region of the goat BLG gene,the sgBLG/Cas9 vector was designed and constructed,and it was electrotransfected goat fetal fibroblasts.The site-mutagenic activity of the BLG gene locus was verified by PCR.Based on BLC14 mammary gland-specific expression vector,three hLF gene targeting vectors with homology arm length(6.0,3.5 and 1.2 kb)were constructed,and it was cotransfected into goat fetal fibroblasts with sgBLG/Cas9 vector respectively.After 500µg/mL G418 screening,the gene targeting was detected by PCR.【Result】The mutagenic activity efficiency of cleaving DNA double strands with BLG locus in goat fetal fibroblasts was 30%-35%by sgBLG/Cas9 vector.The hLF gene targeting vectors(BLC14-1,BLC14-2 and BLC14-3)were constructed to obtain three homology arm lengths,corresponding to 6.0,3.5 and 1.2 kb.Three hLF gene targeting vectors and sgBLG/Cas9 vector were cotransfected into goat fetal fibroblasts.After 5 times of electrotransfections and G418 screening,83,77 and 86 drug-resistant cells were obtained respectively.Finally,42,38 and 44 BLG-/hLF+gene targeting cell lines were obtained by PCR homologous recombination detection.The average targeting efficiencies of hLF gene at the BLG locus of goat fetal fibroblasts were 50.6%(42/83),49.4%(38/77)and 51.2%(44/86).There was no statistically significant difference in the targeting efficiency of hLF gene targeting vectors constructed with three different lengths of homology arms at the BLG gene locus in goats(P>0.05),indicating that the homology arm length had no significant effect on CRISPR/Cas9-mediated hLF gene targeting at the BLG gene locus in goats.【Conclusion】A number of BLG-/hLF+gene targeting cell lines(BLG loci for site-directed target hLF genes)have been successfully obtained by CRISPR/Cas9 system-mediated hLF gene targeting at BLG gene locus in goat fetal fibroblasts.However,the length of the homology arm of the targeting vector has no significant effect on the targeting efficiency of the CRISPR/Cas9 system-mediated BLG site-directed integration of the hLF gene.