Identification of new antibiotic targets using a novel CRISPRi system
Key information
Research topics
This is a summer student position supervised by Brindha Gap-Gaupool in Eachan Johnson's lab.
Introduction to the science
Pathogenic bacteria have evolved resistance to current antimicrobial drugs. Antibiotic-resistant infections killed 1.27 million people in 2019, and this figure is predicted to rise drastically over the next few years. Therefore, we need new antibiotics which bacteria are less likely to develop resistance against. To help us identify new drug targets whose brief inhibition kill the bacteria, we have developed system to transiently knock down expression of any gene.
About the project
We use CRISPR interference (CRISPRi) to knock down espression. CRISPRi uses a catalytically inactive dCas9 directed by a single guide RNA (sgRNA) to block the transcription of target genes, without cutting the DNA. The dCas9-sgRNA complex has a very high affinity for its target DNA which means that transcriptional inhibition cannot be turned off. Therefore, developing an OFF switch would help us identify the gene functions whose transient inhibition causes irreversible death spiral in a bacterium.
To develop this switch, we will exploit the proteolytic activity of Tobacco Etch Virus (TEV) protease which is highly sequence specific. We will tag the N-terminus of the dCas9 with a recognition signal for the TEV protease. Upon TEV activation, dCas9 is cut, exposing a N-degron leading to the removal of the dCas9 and relieving the transcriptional inhibition.
The successful candidate will assist in the testing and validation of our OFF-switch system in Gram-negative bacteria. The student will learn an array of techniques including the handling of Gram-negative bacteria, aseptically and safely. The student will be taught how to clone, from vector and insert preparation to transformation of competent bacteria. To verify their plasmids, student will learn how to prepare DNA samples for sequencing. The student will also undertake phenotypic assays using fluorescent reporter bacterial strains. By the end of their placement, the student will have a good grasp on fundamental molecular techniques.
Candidate background
The post holder should embody and demonstrate the Crick ethos and ways of working: bold, open and collegial. The candidate must be registered at a UK Higher Education Institution, studying in the UK and must have completed a minimum of two years’ undergraduate study in a relevant discipline, and on track to receive a final degree grade of 2:1 or 1. In addition, they should be able demonstrate the following experience and key competencies:
- This project would suit a candidate studying biomedical sciences, biological sciences, biochemistry and microbiology. The successful candidate will have some wet-lab experience i.e. practical classes and have a clear understanding of basic molecular techniques such as cloning and PCR and an interest in infectious diseases and molecular genetics.
- Good knowledge in relevant scientific area(s)
- Good written and spoken communication skills
- Ability to work independently and also capable of interacting within a group
References
1. Peters, J.M., Koo, B.M., Patino, R., Heussler, G.E., Hearne, C.C., Qu, J., . . . Rosenberg, O.S. (2019)
Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi.
Nature Microbiology 4: 244–250. PubMed abstract
2. Ghavami, S. and Pandi, A. (2021)
CRISPR interference and its applications.
Progress in Molecular Biology and Translational Science180: 123–140. PubMed abstract