Fast Genome Editing Of Filamentous Fungi Based On CRISPR System

Fungi have attracted much attention because of its ability of protein secretion.Aspergillus niger is one of common filamentous fungi which produces many enzyme,such as alpha-amylase,pectinase,glucoamylase,glucoamylase,protease,cellulase,tannase,etc.For humans,it will bring great benefits if we can achieve the genome editing of A.niger.The difficulty of fungal transformation limits the genomic editing of filamentous fungi.In recent years,CRISPR(Clustered regularly interspersed short palindromic repeats)has became a burgeoning editing technology owing to its unique characteristic.So far,the CRISPR system less applied in the field of filamentous fungi.In this study,the applicability of CRISPR system in A.niger was explored by attempting to edit the genome of filamentous fungi using CRISPR system.The main research results are as follows:(1)The construction vector of pCAMBIA 1302-hFnCpflhFnCpfl was derived from Francisella novicida,which has endonuclease activity.hFnCpf1 gene was cloned from pcDNA3.1 plasmid with PCR method and ligated to the pCAMBIA1302 vector which carries the hph gene.In this process,no redundant sequences were introduced to ensure the correctness of the hFnCpf1 protein reading framework,and the pCAMBIA1302-hFnCpf1 vector was successfully constructed.(2)The establishment of Agrobacterium-mediated transformation(ATM)system forA.niger CBS513.88By optimizing the seven influencing factors,an efficient transformation system was established.In this transformation system,using 1.2 × 107 spores with hygromycin as the screening label,85 transformants was obtained.The pCAMBIA1302-hFnCpfl was successfully integrated into the genome of A.niger CBS513.88(the hygromycin gene on pCAMBIA1302 vector also was integrated into the genome of A.niger CBS513.88).(3)The design and synthesis of sgRNAConsidering the seed sequence,the target editing site(choosing hygromycin resistance gene as target gene),hairpin structure and PAM(protospacer adjacent motif)sequence,DNA sequence(5 '-AATTAACCCTCACTAAAGAATTTCTACTGTTGT AGATTGCGGCATTGTCCGTCAGGACA-3 ')that are complementary to sgRNA are determined.Subsequently,the sgRNA was obtained by in vitro transcription using the sequence as a template.Compared with the direct synthesis of sgRNA,in vitro transcription is more economical.(4)Verify the applicability of CRISPR/Cpf1 in A.niger CBS513.88.The exogenous sgRNA was transiently introduced into A.niger CBS513.88-pCAMBIA1302-hFnCpf1 by electroporation transformation using the hygromycin gene on the A.niger CBS513.88 genome as target editing gene.The aim mutants were analyzed by the method of screening and labeling with hygromycin.BLAST comparison of 10 mutant sequences was performed to analyze the position of the mutation.And the expression framework of the mutant was analyzed by Clustal X,and the experimental results were discussed.In this study,CRISPR system was applied in genomic editing of A.niger for the first time,and achieved the success of the genome editing.This work supplemented the application of the CRISPR system in filamentous fungi.