Construction And Application Of Plasmid Editing System Based On CRISPR-Cas12a And λ Red Recombinase System

Plasmids are useful tools for studying genetic information in living cells,as well as heterogeneous expression of genes and pathways in cells of bacteria,viruses and eukaryotes cells.Various methods have been developed for plasmid manipulation both in vivo and in vitro such as enzyme digestion and ligation,Gibson cloning.Cloning of genes,and even large gene clusters,is no longer a main obstacle.However,these methods are limited by restriction sites,introduced additional sequences through restriction sites,and DNA sequences which are complex or difficult to amplify beyond a certain length,especially in large plasmids,and the operation is also complex and time-consuming when constructing plasmid libraries.To solve these problems,recombineering-based plasmid editing approaches have been developed that allow construction of plasmids with precise junctions without the requirement for restriction enzyme recognition sites.Recombination is a powerful in vivo technique that uses phage recombinant proteins to promote oligonucleotide or linear DNA-mediated homologous recombination and has been widely used in the genome manipulation of many prokaryotes.In these applications,the method is used to make modifications such as base mutations,deletions,or insertions in plasmids,BACs,and the genome of prokaryotes.Although recombination is a common technique,its relatively low recombination frequency usually requires the selectable markers or the counter-selectable cassettes,which leaving unwanted sequences on the plasmids,or the ability to construct mutations without selectable markers is limited.In addition,multimeric or mixtures of recombinant and parental plasmids are formed when plasmids are modified using recombineering,limiting its application,especially in the case of multicopy plasmids.To solve these problems,recombinant engineering coupled CRISPR-Cas system is a simple and effective way.The RNA-guided CRISPR-Cas endonuclease system,an adaptive immune system of prokaryotes,has recently been developed as an efficient genome editing tool Casl2a(also known as Cpfl)is a class II/type V CRISPR-Cas DNA endonuclease,a dual nuclease possessing activities for crRNA processing and DNA cleavage,recognizes the thymidine-rich PAM protospacer-adjacent motif(PAM)sequence(YTN)and cleaves DNA guided by a single crRNA.CRISPR-Cas 12a has been widely used for genome editing in mammals,plants and bacteria.Our lab developed a CRISPR-Casl2a-assisted recombineering system for genetic manipulation of the genomes of Escherichia coli,Yersinia pestis,and Mycobacterium smegmatis.Here,we developed an in vivo plasmid editing system that combines the advantages of CRISPR-Cas 12a and recombineering.We demonstrate the simplicity and high effectiveness of the resulting system for generating point mutations,deletions,and insertions in pUC-based plasmid without formation of multimers and mixtures.Furthermore,using a BAC-based reverse genetic method for mouse hepatitis virus(MHV),the system achieved 22.5-100%efficiency on point mutations,gene deletions,and insertions.Meanwhile,we showed that chemically competent cells can be efficiently used for plasmid editing using our system.We took the plasmid pBAC-JHMVIA as the target plasmid,and replaced the gene NS4 on the genome of MHV with the gfp with 50 bp homologous arm to generate the plasmid of MHV-JHM-GFP,which not only induced large syncytium formation on DBT cells,but also showed high level of expression of GFP protein as expected.The PAM sequence of Cas9 from thermophilic streptococcus(Cas9sth1)is NNAGAAW,but different PAM sequences might be tolerated.We generated a random PAM candidate pool(7 bp random mutation plasmid library)for Cas9sthi using our plasmid editing system,which contain 80%of possible mutations(13200 vs.47=16384).In summary,we have developed a robust and efficient method to modify various plasmids,thereby facilitating studies in bacteria,viruses,and eukaryotes.The widespread prevalence of drug-resistant tuberculosis has become a major concern in the prevention and control of tuberculosis in China,and it is urgent to develop new effective anti-tuberculosis drugs.Studies have shown that the toxin-antitoxin system,widely distribute in the genome of prokaryotes,regulate the adaptive mutant and promote drug-resistant bacteria and latent infections.There were about 80 pairs of TA in the pathogenic mycobacterium tuberculosis,while as for the non-pathogenic mycobacteria,4 pairs of TA in mycobacterium smegmatis,and only 1 pair of TA in mycobacterium aeruginosa,it is unclear whether they were involved in the formation of drug-resistant strains and the pathogenicity of tuberculosis and we constructed a CRISPRi library targeting TA using the CRISPR-dCas9Sth1 recombineering system.The plasmid library was transferred to mycobacterium tuberculosis to generate silenced libraries that inhibiting different TA genes.After the dCas9Sth1 and sgRNA were induced or uninduced,the cell libraries were treated with anti-TB drugs such as bedoquinoline,capreomycin,delamanid and rifampicin etc.With the analysis of next-generation sequencing(NGS),six couples of TA system Rv 1130c-1102c,Rv1991a-1991c,Rv2654c-2653c,Rv2865-2866,Rv3357-3358 and Rv0059-0060 were screened out.Futher studies on above genes and its mechanism in the formation of drug-resistant bacteria strain will continue.