Reprogramming the endogenous type III-A CRISPR-Cas system for genome editing and RNA interference in Mycobacterium tuberculosis

Purpose: Understanding the functional genomics of M.tb (Mycobacterium tuberculosis) and development of novel anti-M.tb drugs and vaccines needs an efficient gene edit tool. The aim of this study was to describe an easy and efficient gene edit tool for functional genomics study of M.tb. Method: A plasmid was designed containing mini CRISPR array and 400bp up and downside homologues DNA sequences from the target gene interposed by EGFP or BFP. This plasmid was transformed into M.tb that guided the endogenous CRISPR system of M.tb to cut the target gene and insert EGFP or BFP through homologues ends joining. The EGFP/BFP insertion was confirmed and the total genomic was extracted from mutated and wild type strains and subjected to High-throughput DNA sequencing. Results: The raw data was filtered by Trimmomatic and the clean reads were mapped to M.tb H37Ra reference genome with bwa 99.94%, and 99.97% genome of wag31 and esxQ deletion strains were covered respectively. Overall design: DNA library for wild type, H37Ra ?wag and H37Ra ?esxQ was constructed in triplicate usning Nextera Illumina kit and were subjected to high-throughput sequencing.