Functional Characterization Of The Endogenous Type I-F CRISPR-Cas System Of Zymomonas Mobilis And Its Applications In Genome Editing

Zymomonas mobilis is an important industrial microorganism with many attractive physiological attributes as a cell factory.Highly efficient genetic manipulation methods,such as the genome editing technology based on CRISPR-Cas(clustered regularly interspaced short palindromic repeats-CRISPR-associated)systems will further facilitate the research,development and application of this bacterium.The CRISPR-Cas-based technology has attracted extensive attentions as it has enabled rapid,easy and high-throughput microbial engineering,being applied in some model microorganisms,such as Escherichia coli and Saccharomyces cerevisiae.However,this technology has been generally restricted in the most majority of prokaryotes due to the potential cytotoxicity of exogenous CRISPR-Cas to the host cells.CRISPR-Cas systems naturally occur in many prokaryotic microorganisms,among which Type I systems are the most abundant.Such endogenous systems can be exploited as genome editing toolkits for strain development upon demonstrating their functions.The model species Z.mobilis subsp.mobilis ZM4 contains a complete endogenous Type I-F CRISPR-Cas system,which is consisted of 4 CRISPR arrays and 6 cas genes.The cas genes are organized as an operon,including cas1,cas2/3,cas8,cas5,cas7,and cas6.Transcriptional data revealed the constitutive expression of the CRISPR loci and cas genes.In this study,the DNA targeting activity of the endogenous Type I-F CRISPR-Cas system was determined via a plasmid interference assay.Meanwhile,the influence of various trinucleotide combinations on plasmid DNA interference was evaluated by constructing a library of protospacer adjacent motifs(PAMs)with 4 active PAMs identified,which are 5'-ACC-3',5'-TCC-3',5'-CCC-3'and 5'-GCC-3'.These results thus laid a solid foundation for further exploiting the endogenous Type I-F CRISPR-Cas system of ZM4 as a genome editing toolkit.The genome editing toolkit based on the endogenous Type I-F CRISPR-Cas system of ZM4 developed in this study includes a series of genome editing plasmids,each carrying an artificial genome-targeting CRISPR and a donor DNA for facilitating homologous recombination.Through this toolkit,various genome editing purposes were efficiently achieved.Among them,single gene knockout,insertion,replacement,and in situ tagging,as well as nucleotide substitution all reached an editing efficient of 100%,while deletion of a large genomic fragment and multiplex gene deletion attained editing efficiencies of 50% and 18.75%,respectively.It was also demonstrated that the inactivation of a DNA restriction-modification system could improve transformation efficiencies of genome editing plasmids and hence the genome editing efficiency.This study therefore not only provided a powerful genetic manipulation toolkit for Z.mobilis and similar microorganisms,but also might shed light on exploiting endogenous CRISPR-Cas systems in other microorganisms for genome editing,being of great research significances and comprehensive application prospects.