MYCOBACTERIUM TUBERCULOSIS GENOME INDEL AND CRISPRI HIGH- THROUGHPUT SCREENING BY MANIPULATING ITS ENDOGENOUS TYPE IIIA CRIPSR.

Reconnoitering the new genetics tools for identifying novel vaccine candidates and drug targets is the need of time to combat tuberculosis (TB). This will bring forth the comprehension of functional genomics, virulent genes, latency in host and resistance against drugs in the causative agent of TB i.e. Mycobacterium tuberculosis (Mtb). Keeping in view, Mtb endogenous CRISPR-Cas type III a system was exploited to envisage genome editing, mechanism of RNA interference, and throughout Mtb genome CRISPR interference (CRISPRi) screening using in-vitro and in-vivo. A repeat sequence of type IIIA CRISPR array was incorporated in pMV261-crRNA. Guide RNA for the target gene was designed and homology- directed repair (HDR) sequence containing GFP was employed for gene insertion- deletion (indel). For gene knock-down (RNA interference) mechanism was identified by RIP-qPCR. Guide RNA library (5658 in numbers) for the whole genome was designed from Twist Bioscience Company and high-throughput screening was performed for in-vitro and in-vivo growing Mtb using bioinformatics tools. qPCR declared the presence of type III A CRISPR array in Mtb. Using pMV261-crRNA and gRNA for the target gene successfully knocked-in GFP adjacent to gyrA gene and successfully knocked-out lpqE, lpqN, esxA and lpqD separately. For knock-down, katG, esxT, and dcd genes were successfully interfered at transcriptional level using CRISPRi. This was validated by RIP in which csm6-HA tag pull down CRISPR from bacteria having gRNA for katG or dcd or esxT. High-throughput screening analysis showed 228 growth promoting and 385 inhibiting genes in in-vitro culture and 29 growth promoting and 4 inhibiting genes in in-vivo environment. This is your futuristic vision, not a conclusion, please conclude your study in 1 sentence. This system can be used for genetic manipulation, exploring functional genomics of Mtb and identifying new vaccine candidates and drugs targets in Mtb. [ABSTRACT FROM AUTHOR]