CRISPR Adaptation in the human pathogen Mycobacterium tuberculosi

Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb). About one third of the world’s population is infected with TB, and in 2014 approximately 1.5 million people died due to infection, with the burden highest in Africa. Multi-drug resistant (MDR) forms of TB are an increasing concern, and new treatments are required urgently. The lack of good genetic tools has hampered research on Mtb.

The aim of this project is to investigate the CRISPR-Cas system of Mtb, which remains completely uncharacterised, to determine its function in the organism and its utility in the genetic manipulation of Mtb and the wider study of gene expression.

CRISPR stands for “Clustered Regularly Interspaced Short Palindromic Repeats”. These direct repeats were observed in the E. coli genome in 1987 and are present in many prokaryotes. Subsequently it was recognised that the regions between the repeats, called spacers, often matched viral and plasmid sequences. New spacers are captured during viral infection by a poorly understood process known as Adaptation, which involves the Cas1 and Cas2 proteins. Spacers are transcribed and direct the CRISPR effector complexes to target and destroy cognate nucleic acid sequences.

Mtb has a complete CRISPR-Cas system including a CRISPR array close to a Type IIIA (CSM) effector system and a copy of Cas1 and Cas2 for adaptation. Although all the necessary components are present the system has not been studied yet. We will investigate the activity of the Mtb Cas1 and Cas2 proteins using assays developed in our laboratory to assess the potential for integration of defined DNA sequences at the chromosomal CRISPR site in Mtb, initially in vitro. If we can use the CRISPR system to introduce specific DNA sequences this would open the door to programmed targeting of selected genes to control gene expression, which would be a very useful addition to our toolkit for the fight against TB.