A Study Of Single-base Gene Editing In Mycobacterial Genomes

Genus Mycobacterium,including Mycobacterium tuberculosis and Mycobacterium abscessus,are still important pathogens that threat public health.According to statistics,about 1.5 million people died of tuberculosis(TB)worldwide in 2018,with 10 million people developing TB.Therefore,it is very important to understand pathogenic and drug-resistant genes of Mycobacterium tuberculosis,and then to understand the related mechanisms.Single base editing is a form of genome editing that enables direct,irreversible conversion of one base pair to another at the target gene locus without requiring double-stranded DNA breaks(DSBs)and other repair methods such as non-homology end joining.In this study,we combined adenosine deaminase,cytidine deaminase with dCas9 protein separately to construct the single base editor,which can convert genomic bases A to G by adenosine deaminase base editor,or mediate genomic base C to T conversion by cytidine deaminase base editor.We have achieved precise editing of single base in the genome of Mycobacterium smegmatis or Mycobacterium tuberculosis,and successfully realized the exchange of A:T and C:G base pairs.What's more,the repairing-related Rec A protein and NucS protein affect the occurrence of single-base editing.The efficiency of single-base editing can be improved when the function of this protein is inhibited.We can realize precise gene editing and the efficient construction of traceless mutations or gene knockout strains.Next,we will optimize this system to improve the efficiency of single-base editing in Mycobacterium tuberculosis to provide support for the further study of Mycobacterium tuberculosis.