多靶点CRISPR表达载体系统的设计和构建

Design and Construction of CRISPR Expression Vector System Harboring Multi-Target Sites

CRISPR/Cas9核酸酶系统能实现多基因的高效靶向编辑,但是多靶点CRISPR表达载体的构建比较复杂。本研究以细菌的DNA旋转酶和二氢叶酸还原酶为靶基因,选择多个打靶位点,合成携带靶序列和多克隆位点的寡聚核苷酸引物,直接退火延伸合成靶序列DNA,利用同尾酶产生相同粘性末端的特点,将靶序列克隆至骨架载体,然后依次加入更多靶序列,实现多靶点的串联。最后测序验证,单靶点、双靶点和三靶点成功插入CRISPR表达载体,同时保留了多克隆位点,实现多靶点串联。本研究设计的多靶点CRISPR表达载体系统操作简单、成本低、成功率高,为后续细菌多基因编辑提供技术支撑,同时可拓展应用于其他生物多基因打靶研究。

CRISPR/Cas9 nuclease system has the ability to efficiently target multiple genes simultaneously, but the construction of CRISPR expression vectors containing multi-target sequences is complicated. In this study, bacterial DNA gyrase and dihydrofolate reductase were chosen as target genes. Multiple targeting sites were selected to synthesize oligonucleotide primers carrying the target sequence and multiple cloning sites. Then, dimers of two oligonucleotides were extended via Klenow enzyme to obtain target DNA sequences. Based on characteristics of isocaudarner, target DNA fragments were cloned into backbone vector. After that, much more target sequences were inserted into vector one by one to achieve vectors with multiple targets after sequencing. At last, sequencing verification showed that single target, double targets and triple targets were successfully inserted into CRISPR expression vector, meanwhile multiple cloning sites were remained to achieve multi-target series. In conclusion, the CRISRP expression vector system designed in current study was feasible, low-cost and of high success rate. which provided technical support for follow-up multiple bacterial genes editing and expanded the application to study other biological genes targeting.