Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase.

Xin-Yuan Ding,Guo-Bao Li,Guo-Liang Zhang,Yi-Cheng Sun,Yi-Man Geng,Si-Shang Li,Mei-Yi Yan

doi:10.3389/fgeed.2021.734436

Abstract

Multidrug-resistant Mycobacterium tuberculosis (Mtb) infection seriously endangers global human health, creating an urgent need for new treatment strategies. Efficient genome editing tools can facilitate identification of key genes and pathways involved in bacterial physiology, pathogenesis, and drug resistance mechanisms, and thus contribute to the development of novel treatments for drug-resistant tuberculosis. Here, we report a two-plasmid system, MtbCBE, used to inactivate genes and introduce point mutations in Mtb. In this system, the assistant plasmid pRecX-NucSE107A expresses RecX and NucSE107A to repress RecA-dependent and NucS-dependent DNA repair systems, and the base editor plasmid pCBE expresses a fusion protein combining cytidine deaminase APOBEC1, Cas9 nickase (nCas9), and uracil DNA glycosylase inhibitor (UGI). Together, the two plasmids enabled efficient G:C to A:T base pair conversion at desired sites in the Mtb genome. The successful development of a base editing system will facilitate elucidation of the molecular mechanisms underlying Mtb pathogenesis and drug resistance and provide critical inspiration for the development of base editing tools in other microbes.

Highlights

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and the leading cause of death from a single pathogen
Mtb strains H37Ra, M. smegmatis strain mc2 155, and their derivatives were used in this study; all strains are listed in Supplementary Table S1
To determine whether deaminase-mediated targeted mutagenesis could be achieved in M. smegmatis, we constructed base editor plasmids expressing a fusion protein consisting of dead Cas9 or nCas9 from Streptococcus thermophilus (Rock et al, 2017), cytidine deaminase (APOBEC1) at the N-terminus, and uracil DNA glycosylase inhibitor (UGI) at the C-terminus under the control of the anhydrotetracycline (ATc)-inducible PtetO promoter; the plasmids expressed an sgRNA cassette under the control of a constitutive promoter (Figure 1A)

Summary

Introduction

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and the leading cause of death from a single pathogen. The World Health Organization (WHO) estimated that, in 2019, 10 million new patients around the world were diagnosed with TB (World Health Organization, 2020). The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains is an ongoing health problem that creates an urgent need for novel therapeutic strategies. Identification and characterization of drug targets strongly rely on efficient genetic manipulation techniques. Genetic manipulation of mycobacteria is challenging, mainly due to their slow growth, the pathogenicity of some species, and their GCrich genomes. Several techniques for genetic engineering have been developed in mycobacteria (Chhotaray et al, 2018; Murphy et al, 2018), benefiting especially from the advent of the Programmable Base Editing in Mtb

Methods

Results

Conclusion