利用CRISPR-Cas9技术敲除MAS1基因的质粒构建与酶切方案优化的研究

Construction and Optimization of Enzyme-Cutting Scheme by Using CRISPR-Cas9 Technology to Knockout the MAS1 Gene

CRISPR-CAS9技术是新一代基因编辑技术,可简便快捷地对哺乳动物细胞进行指定基因的敲除,实现沉默外源基因表达的目的。但传统的CRISPR-CAS9技术提供的酶切方案没有特异性,致使酶切效率不稳定,影响胶回收率以及后续实验。本实验通过改变酶切体系大小,以及酶切反应时间,继而通过胶回收率及测序检测的方法验证最佳酶切条件,最后通过荧光定量PCR和Western blotting实验检测目的基因的表达量,确认是否成功敲除目的基因。研究结果说明:BbsⅠ酶与px459质粒比值为1μL:500 ng,酶切体系为50μL,酶切时间为45 min时,酶切效果最佳、胶回收率最高,且此时目的基因在细胞内的表达量为0,确定该实验条件下目的基因被成功敲除。

CRISPR-CAS9 technology is a new generation of gene editing technology,which can be convenient in mammalian cells for gene knockout.Make the expression of exogenous gene silencing.But the traditional CRISPR-CAS9 technology scheme has no specific restriction,so that the digestion efficiency is not stable.Therefore,affect the inefficiency of glue recovery and subsequent experiments.This experiment by changing the size of enzyme cutting system and the time of enzymatic reaction,then the optimum enzyme digestion conditions were confirmed by the method of gel recovery and sequencing,finally,fluorescent quantitative PCR and Western Blot were used to detect the expression of the target gene,and to confirm whether the target gene was successfully knocked out.The tests results show that:When the proportion of BbsⅠenzyme to px459 plasmid was 1μL:500 ng,and the enzyme digestion system was 50μL.The time of enzyme digestion was 45 minutes,the effect of enzyme digestion was the best,and the yield of gel was the highest,and the expression of target gene in the cells was 0,determine that the target gene under the conditions of the experiment were knocked out successfully.