A CRISPR/Cas9-based visual toolkit enabling multiplex integration at specific genomic loci in Aspergillus niger  

作　　者：Yangyang Li Cen Li Yishan Fu Quan Zhang Jianing Ma Jingwen Zhou Jianghua Li Guocheng Du Song Liu 

机构地区：[1]National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi,214122,China [2]Science Center for Future Foods,Jiangnan University,Wuxi,214122,China [3]School of Biotechnology,Jiangnan University,1800 Lihu Road,Wuxi,214122,China [4]Jiangsu Provisional Research Center for Bioactive Product Processing Technology,Jiangnan University,1800 Lihu Road,Wuxi,Jiangsu,214122,China [5]Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),College of Life Sciences/Institute of Agro-bioengineering,Guizhou University,Guiyang,550025,China [6]Dalian Research Institute of Petroleum and Petrochemicals,SINOPEC,Dalian,116000,China [7]School of Chemical Engineering,Dalian University of Technology,Dalian,116000,China

出　　处：《Synthetic and Systems Biotechnology》2024年第2期209-216,共8页合成和系统生物技术（英文）

基　　金：This work was financially supported by the National Key Research and Development Program of China(2019YFA0706900);the National Natural Science Foundation of China(No.32071474);the Postgraduate Research and Practice Innovation Program of Jiangsu Province(KYCX20_1821).

摘　　要：Aspergillus niger is a highly versatile fungal strain utilized in industrial production.The expression levels of recombinant genes in A.niger can be enhanced by increasing the copy number.Nevertheless,given the prolonged gene editing cycle of A.niger,a“one-step”strategy facilitating the simultaneous integration of recombinant genes into multiple genomic loci would provide a definitive advantage.In our previous study,a visual multigene editing system(VMS)was designed to knock out five genes,employing a tRNA-sgRNA array that includes the pigment gene albA and the target genes.Building upon this system,hybrid donor DNAs(dDNAs)were introduced to establish a clustered regularly interspaced short palindromic repeats(CRISPR)-based multiplex integration toolkit.Firstly,a CRISPR-Cas9 homology-directed repair(CRISPR-HDR)system was constructed in A.niger by co-transforming the CRISPR-Cas9 plasmid(with a highly efficient sgRNA)and the dDNA,resulting in precise integration of recombinant xylanase gene xynA into the target loci(theβ-glucosidase gene bgl,the amylase gene amyA,and the acid amylase gene ammA).Subsequently,the length of homology arms in the dDNA was optimized to achieve 100%editing efficiency at each of the three gene loci.To achieve efficient multiplex integration in A.niger,the CRISPR plasmid pLM2 carrying a sgRNA-tRNA array was employed for concurrent double-strand breaks at multiple loci(bgl,amyA,ammA,and albA).Hybrid dDNAs were then employed for repair,including dDNA1-3(containing xynA expression cassettes without selection markers)and dDNAalbA(for albA knockout).Among the obtained white colonies(RLM2′),23.5%exhibited concurrent replacement of the bgl,amyA,and ammA genes with xynA(three copies).Notably,the xynA activity obtained by simultaneous insertion into three loci was 48.6%higher compared to that obtained by insertion into only the bgl locus.Furthermore,this multiple integration toolkit successfully enhanced the expression of endogenous pectinase pelA and Candida antarctica lipase CALB.Hence,the

关 键 词：Multiplex integration Aspergillus niger CRISPR-Cas9 homologous direct repair White colony 

分 类 号：Q78[生物学—分子生物学]