Reprogramming The Endogeneous Type ?-A Crispr-Cas System For Genome Engineering In Mycobacterium Tuberculosis

Tuberculosis(TB)is a leading infectious disease of the 21^th century.The disease is caused by gram-positive,rod-shaped bacillus known as Mycobacterium tuberculosis(Mtb).About one-third population of the world is latently infected showing extensive reactivation and spread of the disease.Antibiotic resistance to anti-tuberculosis drugs is a hindrance to effective TB prevention and control globally.Currently,the only one available vaccine for TB is BCG discovered in the early last century.The major drawback of this vaccine is its failure to prevent pulmonary TB in adolescents.Moreover,in some cases vaccine causes infection in immunocompromised children.The cost-effective vaccine works effectively against those pathogens which are cleared by humoral immunity.While in TB infection cellular arm provides immunity so the development of vaccine driving the cellular immunity will have more prophylactic effects.The development of a vaccine is based on gene deletion thus making the pathogen avirulent.So far targeted genetic manipulation in Mtb is done by recombination-based tools.However,these tools are not widely applicable due to high cost and time consumption,low efficiency and specificity.Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)and CRISPR-associated proteins(CRISPR-Cas9)based genome editing in several organisms has opened up research avenues for effective vaccine production against several pathogens.CRISPR-Cas system is classified into several types and their respective subtypes.The presence of type ?-A CRISPR-Cas system led us to investigate the system for genome engineering purposes in Mtb.In this study,we reprogrammed the endogenous type ?-A CRISPR-Cas system for efficient gene knock-in by the simple delivery of plasmid expressing the mini-CRISPR assay,and a donor DNA template.We achieved an efficient EGFP insertion into the genome.We applied this system for gene deletion;ten autophagy-related genes(lipoprotein and early secretary protein family)were knocked out with a high efficiency without off-targeting as observed by deep sequencing.In addition,we investigated silencing of genes by selecting 40 bp DNA sequence flanked by a pentanucleotide motif5‘-GAAAC-3‘in the non-coding sequence of a gene that would only target the transcript but not DNA.In agreement,we found a significant and high efficiency(60-90%)downregulation of five genes.Finally,we investigated multiple spacers targeting the mRNA of three genes lpq E,kat G and inh A,reduced their mRNAs level leading to 60-70%downregulation simultaneously.Our work reports a novel potent tool to edit any locus with high efficiency and specificity and gene silencing of several including growth essential genes in Mtb.The deletion and phenotypic analysis of genes involved in cell division in this study further demonstrated the significance of this approach to gain further insights into gene functions.The simultaneous gene silencing might overcome the problem of cross-resistance arise during conventional gene silencing due to an introduction of marker antibiotic resistance genes.Thus type ?-A CRISPR system provides an efficient approach to edit genes for the development of the prophylactic vaccine.An important research area will be to gain an insight into the molecular mechanism of target recognition and its destruction.