Keywords

CRISPR Interference; mycobacteria; antibiotic target discovery; microbiology; reporter system; molecular biology

Abstract

Among nontuberculous mycobacteria (NTM), Mycobacterium abscessus (Mab) poses a particular threat to patients with cystic fibrosis and chronic obstructive pulmonary disease due to the lung damage and higher mortality rate it causes. Current treatments involve prolonged multi-antibiotic regimens that are often ineffective, highlighting the need for new drug targets.

The CRISPR Interference (CRISPRi) system uses a deactivated Cas9 protein (dCas9) to enable inducible gene silencing, offering a potential approach to identifying new targets. Genes that lead to a loss of Mab viability when silenced are considered essential, an important attribute of potential drug targets. While CRISPRi has shown promise in Mycobacterium tuberculosis, its use in Mab has been less successful. This project seeks to optimize CRISPRi functionality in Mab, ultimately enabling genetic screening to identify novel drug targets. More specifically, this project’s goals include studying the effects of modifying the length of single guide RNA (sgRNA), which helps the dCas9 protein find a gene of interest, and testing the strength of different protospacer adjacent motif (PAM) sequences, which affect dCas9 binding.

This project also worked to develop part of a luciferase reporter system to visualize cell death and confirm gene silencing through the loss of luminescence. When the CRISPRi component of the transcriptional reporter is complete, it should help detect false-negative results, helping to identify cases where Mab survives due to ineffective CRISPRi silencing, rather than because the targeted gene is nonessential. By comparing cell death to the amount of CRISPRi-mediated silencing, gene vulnerability can be assessed, aiding in the identification and prioritization of potential antimicrobial drug targets.

Thesis Completion Year

2025

Thesis Completion Semester

Spring

Thesis Chair

Rohde, Kyle

College

College of Medicine

Department

Burnett School of Biomedical Sciences

Thesis Discipline

Biomedical Sciences

Language

English

Access Status

Open Access

Length of Campus Access

None

Campus Location

Orlando (Main) Campus

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Microbiology Commons

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Rights Statement

In Copyright