Preliminary Study On The Establishment Of Crispr/Cas9 Genome Editing System Of Metarhizium Robberstii

Entomogenous fungi is a kind of fungus parasitic on insects and causing the death of insects,which is widely used in agricultural production for pest control because of its strong pathogenicity to pests,rapid propagation of insect pests and high environmental friendliness.Metarhizium robberstii is a broad-spectrum insecticidal fungus,it is strong in pathogenicity,harmless to crops and easy to mass production.However,the genetic transformation of Metarhizium robberstii has been limited by the low homologous recombination efficiency factors.While the nucleic acid enzymes represented by zinc finger nucleic acid enzyme and talen can achieve the goal of genome editing,but the cost is too high,the operation process is complicated,it is difficult to achieve fast and efficient editing of genome.CRISPR/Cas9 is a popular research in recent years,it has the characteristics of simple operation,low cost and so on,so the efficient editing of the Metarhizium robberstii genome is an achievable goal.The aim of this study was to establish an effective CRISPR/Cas9 genome editing system in Metarhizium robberstii,to introduce Cas9 protein sequence and SgRNA sequence into Metarhizium robberstii through Agrobacterium tumefaciens transformation,and to test the effect of its editing function,mainly achieved the following results:(1)The Cas9 protein sequence was obtained from the literature of CRISPR/Cas9 system,which was constructed by the Metarhizium robberstii,and the Cas9 protein sequence was optimized according to the gene sequence of the Metarhizium robberstii.(2)The Cas9 protein sequence is introduced into Metarhizium robberstii in two steps,the SgRNA sequence was then introduced,and the target sites were located in the multiple copper oxidase gene of the Pseudomonas aeruginosa,and the results of the inversion showed that the Cas9 protein sequence was successfully transcribed into mRNA,but the phenotype and sequencing results of the transformed children showed the CRISPR/ Cas9 system did not edit the target DNA sequence.(3)We improved the two-step construction of CRISPR/Cas9 system,the Cas9 protein and SgRNA sequence are connected to a plasmid,the target sites are in the green fluorescent protein sequence on the plasmid,then the plasmid is introduced into the Metarhizium robberstii.The protein imprinting test of the strain of the imported plasmid showed that the Cas9 protein was successfully expressed,and the green fluorescent proteincould be observed by fluorescence microscopic observation of the strains under ultraviolet.It is indicated that the CRISPR/Cas9 system does not cut the target sites of green fluorescent protein gene.The CRISPR/Cas9 gene editing system constructed by the Metarhizium robberstii did not edit the target gene loci successfully,but the presence of Cas9 protein in the Pseudomonas aeruginosa cells was validated.Following the analysis of the U6 promoter sequence of SgRNA and the comparison with the green strain sequence,the U6 promoter sequence in the sequence of Roberts Green strains was found.Subsequent experiments can replace the U6 promoter in the bar plasmid of one step construction method with the U6 promoter of the internal source of Metarhizium robberstii.This study combined with the CRISPR/CAS9 gene editing system aims to establish an efficient,fast and easy to operate genomic editing technique in Metarhizium robberstii based on the current research.Although the system has not been successfully constructed,it has laid a solid foundation for the establishment and further improvement of the CRISPR/CAS9 gene editing system of Metarhizium robberstii.