中国鹅掌楸纤维素合酶的CRISPR/Cas9基因敲除系统的gRNA表达载体构建

Expression Vector Construction of Cellulose Synthase gRNA With CRISPR/Cas9 Gene Knockout System of Liriodendron chinense

CRISPR/Cas系统最早发现于细菌,对入侵的病毒或噬菌体DNA具有免疫作用。基于CRISPR/Cas的免疫机制,设计开发的CRISPR/Cas9基因编辑系统已经成为真核生物基因编辑的主流工具,在拟南芥、水稻等高等植物的基因组功能研究中已经广泛应用。本研究以中国鹅掌楸纤维素合酶(Cellulose synthase,CESA)为研究对象,首次构建了具有表达中国鹅掌楸纤维素合酶(Cellulose synthase,CESA)gRNA(guide RNA)的CRISPR/Cas9基因敲除系统,以LcCESA1基因外显子5'端的第一个外显子区域的GN19NGG序列设计gRNA引物;以p201N-Cas9为载体,Gibson Assembly誖Master Mix为连接酶,通过Gibson Assembly连接技术,将CESA1-gRNA连入p201N-Cas9中,获得p201N-Cas9-CESA1-gRNA载体。根据载体中插入序列,设计了两对检测引物,经PCR鉴定,均获得了相应大小的序列;测序分析进一步证实确认gRNA已经完整准确地连入载体。p201N-Cas9-CESA1-gRNA载体能对CESA1基因进行定向编辑,对后续研究鹅掌楸纤维素合酶的基因功能具有重要意义。

The CRISPR/Cas system, firstly discovered in bacteria, has immune effect in the invading virus or the DNA of phage. Based on the immune mechanism of CRISPR/Cas system, the designed CRISPR/Cas9gene-editing system has been widely used in the study of genome function in higher plants such as Arabidopsis and rice, and becomes the main tool for gene editing in eukaryotes. By using the Cellulose synthase（CESA） of Liriodendron chinense as the research object, we generated a CRISPR/Cas9 gene knockout system to express CESA-gRNA（guide RNA）. The gRNA primers were designed using the GN19 NGG sequence of the first exon region at the 5＇ end of the exon of the LcCESA1 gene. Using p201N-Cas9 as the vector, Gibson Assembly Master Mix as the ligase, we connected the CESA1-gRNA to the p201N-Cas9 to construct p201N-Cas9-CESA1-gRNA vector through Gibson Assembly technology. According to the sequence inserted in the vector, we designed two testing primers. The sequences with appropriate size were identified through PCR amplification. With the sequencing result of the insertion sequence, it was confirmed that gRNA has been connected to the vector completely and correctly. The p201N-Cas9-gRNA vector, editing CESA gene directionally, has great significance on confirming the function of cellulose synthase in Liriodendron chinense.