基于CRISPR/Cas9的蛹虫草无抗性标记转化技术的构建

Construction of the CRISPR/Cas9-based marker-free transformation of Cordyceps militaris

传统的真菌遗传改造方法需要抗性标记,但目前可使用的抗性标记基因非常有限,导致蛹虫草遗传改造面临着抗性基因数量不足的问题,且尚未能实现多个目的基因的连续敲入或敲除,因此在蛹虫草中建立高效的无抗性标记转化技术显得尤为重要。本研究利用CRISPR/Cas9技术对蛹虫草的Cmura5基因进行编辑,通过内源5S-1、5S-2和U6启动子对gRNA进行转录,结果表明使用U6启动子对Cmura5基因的编辑效率达到了100%。在尿嘧啶缺陷型菌株Cmura5–中,回补野生型Cmura5基因可实现正向选择,即野生型菌株可以在基础培养基上生长。利用设计的同源臂对Cmura5基因进行回收,可以实现反向选择,即野生型在含有5-氟乳清酸培养基中生长受到抑制。以尿嘧啶缺陷型Cmura5–为出发菌株,利用无抗性标记转化技术,导入一个重组质粒效率为75%;连续导入2个重组质粒效率为80%;连续导入3个重组质粒效率为100%;连续导入4个重组质粒效率为50%,平均转化效率为75.7%,每一轮的标记回收率均在100%,实现了4个外源基因在蛹虫草中同时表达。

Traditional fungal genetic modification methods require resistance markers,but few marker genes are available.The genetic modification of Cordyceps militaris is faced with the problem of insufficient number of resistance genes,while continuous knock-in or knock-out of multiple target genes has not been achieved,therefore,it is particularly important to establish an efficient non-resistant marker transformation technology for C.militaris.In this study,the CRISPR/Cas9 technique was adopted to edit the Cmura5 gene of C.militaris and to transcribe gRNA under the control of endogenous 5S-1,5S-2,and U6 promoters.The result showed that the editing efficiency of the Cmura5 gene using U6 promoters reached 100%.In the uracil auxotroph strain Cmura5–,the rescued wild Cmura5 gene enables forward selection,i.e.wild strains can grow on the basal medium.The Cmura5 gene can be recycled using the designed homologous arm,which allows reverse selection,i.e.the growth of wild type strains are inhibited in mediums containing 5-fluoroorotic acid.Based on the uracil auxotroph strain Cmura5–,a recombinant plasmid was imported by non-resistant marker transformation,and the efficiency of import reached 75%;the efficiency of continuous import of two recombinant plasmids reached 80%;the efficiency of continuous import of three recombinant plasmids reached 100%;the efficiency of continuous import of four recombinant plasmids reached 50%,the average conversion efficiency was 75.7%,and the recovery rate of each round was 100%,accomplishing the simultaneous expression of four exogenous genes in the C.militaris.