基于CRISPR系统的双孢蘑菇基因编辑体系建立及应用

Establishment of a CRISPR/Cas9 System in Agaricus bisporus

根据CRISPR(clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated)基因编辑技术,设计AbPPO4特异性导向RNA,构建双孢蘑菇(Agaricus bisporus)基因编辑载体pAbGEB1-AbPPO4,再通过农杆菌介导转化双孢蘑菇菌丝体建立基因编辑体系,观察菌丝生长状况。结果表明:获得12株转化子,1号和7号的菌丝体中存在潮霉素抗性基因,且AbPPO4基因的相对表达量较低,分别为0.28±0.04和0.47±0.02,与对照组相比,组间差异均具有统计学意义(P<0.05),说明1号和7号转化子的菌丝体AbPPO4基因表达受到了抑制;与对照组相比,1号和7号转化子的菌丝体中AbPPO4基因均发生了突变,1号和7号分别产生了4 bp和11 bp的碱基缺失,证明设计的CRISPR/Cas9系统在双孢蘑菇内对AbPPO4基因成功地进行了编辑;同时发现AbPPO4基因突变后对双孢蘑菇菌丝体的生长产生了不利影响。

A gene editing system based on CRISPR/Cas9 was established in Agaricus bisporus.First,a guide RNA specific to AbPPO4 was designed and vector pAbGEB1-AbPPO4 carrying the hygromycin resistence gene was constructed,and then the vector was transformed into A.bisporus by Agrobacterium-mediated transformation.Transformants were first screened for hygromycin resistency on PDA medium and then confirmed by detection of the hygromycin resistence gene with PCR.The results showed that transformant No.1 and No.7 contained the hygromycin resistence gene and their AbPPO4 expression levels were(0.28±0.04)and(0.47±0.02),respectively.The AbPPO4 expression level of both transformants were statistically lower compared with that of the original strain.There were 4 bp and 11 bp deletions in the AbPPO4 locus of transformant No.1 and No.7,respectively.Compared with the starting strain,both transformant No.1 and No.7 showed weaker mycelial growth,suggesting that disruption of AbPPO4 inhibited mycelial growth.The success of establishing CRISPR/Cas9 gene editing system in A.bisporus offers a new tool for genetic breeding and understanding molecular mechanisms underlying physiological processes of A.bisporus.