Utilizing The CRISPR/Cpf1 System In Pseudomonas Aeruginosa For Genome Editing

Pseudomonas aeruginosa is a prevalent opportunistic pathogen widespread in the environment and is an important model organism to study the characteristics of Gram negative bacteria,such as quorum sensing,biofilms,virulent and drug resistance.Traditional genetic manipulation methods in P.aeruginosa are still time consuming and laborious.Therefore,the development of versatile and feasible genetic tools in P.aeruginosa strains will contribute to the biological and physiological basic research of P.aeruginosa.CRISPR/Cas system,derived from the immune defense system within bacteria and archaea,has been rapidly engineered for genome editing in a variety of organisms.Among them,the novel CRISPR effector protein Cpf1 and the CRISPR/Cpf1 based methods derived from it have further provided more alternative gene editing target site,and they are more simple,accurate and have a low off-target effect,which has attracted wide attention.In this study,it was found that the Fn Cpf1 protein derived from Francisella novicida has high cleavage activity in P.aeruginosa PAO1,which indicated that the activity of endogenous non-homologous end joining related protein and endogenous recombinase were too weak to repair the DNA double-strand breaks caused by Fn Cpf1.Based on this,we subsequently constructed a two-plasmid CRISPR-Cpf1 system,consisting of plasmid pCpf1-?Red and p Cr RNA.The plasmid pCpf1-?Red contains constitutively expressed FnCpf1 protein and L-arabinose-inducible expressed ?Red recombinase.The plasmid pCrRNA contained constitutively expressed the crRNAs,the homology arms for recombination and the sucrose-sensitive counter-selection marker gene sac B to cure plasmid.In terms of gene deletion,the deletion efficiency of the system for pyocyanin production and virulence-related gene phzM(1,013 bp)and membrane protein-related genes oprM(1,461 bp)and mex A(1,178 bp)were 75-100%.In terms of gene insertion and replacement,the replacement efficiency of this system for foreign reporter proteins rfp(783 bp)and lac Z(3,149 bp)is 67-100%,and the insertion efficiency is 75-100%.In terms of the deletion of large DNA fragments,when using the strategy of two tandem cr RNAs,the deletion of phz MS(9.4 kb)and mex Hphz S(14.8 kb)gene clusters associated with pyocyanin production were achieved,with a deletion efficiency of 75%.Subsequently,through substituting p Cr RNA series plasmids,the consecutive deletion of the gene clusters phzA1G1(6.4 kb)and phz A2G2(6.5 kb)related to pyocyanin production was successfully achieved.Finally,the growth curve test showed that the growth rate of the edited mutant strain was not significantly different from the wild-type PAO1 strain.Colony color experiments showed that,for the mutants deleted the genes and gene clusters related to pyocyanin production,there were obvious reduction in pyocyanin except for the ?phz A2G2 mutant.Based on the CRISPR/Cpf1 system,this study successfully established an efficient and rapid gene editing technology in P.aeruginosa,and extended the application of CRISPR/Cas system in P.aeruginosa,which is of great significance for the biological and physiological basic research of P.aeruginosa.