Differential vulnerability CRISPRi screen in Rif Sensitive and Rif Resistant strains of M. tuberculosis

A genome-wide CRISPRi library (RLC0011) was transformed into an M. tuberculosis bioA deletion strain (Rif Sensitive) and an M. tuberculosis rpoB S450L bioA deletion strain (Rif Resistant). The libraries were serially passaged in triplicate in either the presence (CRISPRi on) or absence (CRISPRi off) of ATc. Genomic DNA was harvested at the following generations (cumulative) and subjected to deep sequencing: 0, 2.5, 5, 7.5, 10, 15, 20, 25, and 30. Genetic vulnerability was then calculated, similar to what was described in Bosch & DeJesus et al. (PMID: 34297925). Differential vulnerability of each gene in the S450L mutant strain was calculated with respect to the parental, wild-type M. tuberculosis strain.

本研究将编号为RLC0011的全基因组CRISPR干扰（CRISPRi）文库，分别转化至结核分枝杆菌（Mycobacterium tuberculosis）bioA基因缺失菌株（利福平敏感型），以及rpoB S450L位点突变且bioA基因缺失的结核分枝杆菌菌株（利福平耐药型）。将上述文库分别在添加ATc（CRISPRi激活）或不添加ATc（CRISPRi沉默）的条件下，进行三次生物学重复连续传代。分别在下列累计传代代数收集基因组DNA：0、2.5、5、7.5、10、15、20、25和30代，随后对其进行深度测序。基因脆弱性的计算方法参照Bosch与DeJesus等人发表于PMID: 34297925的研究。本研究以亲本野生型结核分枝杆菌菌株为参照，计算S450L突变菌株中各基因的差异脆弱性。