Construction And Application Of CRISPR-Cas Assisted Genome Editing System In Mycobacteria

Tuberculosis(TB)is an important infectious disease threatening human health.The 2018 global TB report shows the best estimate is that 10 million people developed TB disease worldwide in 2017,with an estimated 1.3 million deaths.Meanwhile,drug-resistant TB continues to be a public health crisis.The best estimate is that,worldwide in 2017,558000 people(range,48300-63900)developed TB that was resistant to rifampicin(RR-TB),the most effective first-line drug,and of these,82%had multidrug-resistant TB(MDR-TB).Among cases of MDR-TB in 2017,8.5%were estimated to have extensively drug-resistant TB(XDR-TB).Therefore,it is particularly important to understand the drug resistance mechanism of Mycobacterium tuberculosis,find related genes involved in drug resistance,and develop new anti-tuberculosis drugs to improve the treatment effect of MDR-TB/XDR-TB.Currently,the study of genes in Mycobacterium tuberculosis has been limited by the lack of effective genetic manipulation techniques to construct gene knockout mutant and point mutant strains.Genetic approaches using nonreplicating vectors,long linear DNA fragments,recombineering,specialized phage transduction have been developed to manipulate M.tuberculosis,but they usually yield low numbers of mutants and require multiple steps to get markerlelss mutants.The low efficiency of these approaches might be related to its extremely slow growth rate(doubling time B24 h).CRISPR-Cas(clustered regularly interspaced short palindromic repeat and CRISPR-associated proteins)system,including CRISPR-Cas9 and CRISPR-Cas 12a(Cpfl),have been widely used as genome editing tools.CRISPR-Cas systems generate highly specific double-strand break(DSB)at the target site,which can be repaired by non-homologous end-joining(NHEJ)or homology repair(HR)pathway,which have brought revolutionary breakthrough for desired genome editing and regulation.The aim of this research was to develop genomic editing tools based on widely used CRISPR-Cas technique suitable for Mtb.The main results were divided into two parts.The first part was to use the ability of DSB generated by CRISPR-Cas,especially CRISPR-Cas 12a to screen the mutant recombinants,while the wild type could be eliminated.First,induced expressionof Cas12a did not strongly affect the growth of M.smegmatis and the cleavage activity of optimized CRISPR-Cas 12a system was validated.Here,we developed and optimized a CRISPR-Cas 12a-assisted recombineering system to facilitate genetic manipulation in bacteria.Using this system,point mutations,deletions,insertions,and gene replacements can be easily generated on the chromosome and it is an efficient approach for generating markerless and scarless mutations in M smegmatis.The second part of this research was to explore the application of CRISPR-Cas assissted NHEJ with one-step deletion in mycobacteria.First,we used Cas12a-assisted recombineering system in Mycobacterium marinum.Interestingly,the results showed that HR did not occur,but the repair of NHEJ occurred.In order to facilitate genome editing in other mycobacteria,we increased the NHEJ activity by three steps:1)Highly expression of.marinum NHEJ machinery;2)repression of RecA-dependent HR;3)generation DSB in stationary phase.We constructed a series genome editing systems in which only the first step was enough to achieve genome editing in Mycobacteria marinum,while two steps were used to achieve genome editing in Mycobacterium smegmatis and three steps were used to achieve efficient genome editing in Mycobacterium tuberculosis.In addition,this system could generate precise deletion and simultaneous double gene mutations in M.tuberculosis.Given to the high efficiency of this Cas9 assisted NHEJ repair system,this system could be used for generation mutant library,which will provide reliable technical support for further study of the function of genes in M.tuberculosis and further understanding of the related mechanisms of drug resistance.