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Abstract

Background

Bacteria and archaea have developed a number of strategies to keep the influx of mobile genetic elements (MGEs) such as viruses and plasmids in check. CRISPR-Cas systems provide adaptive immunity; all types of CRISPR-Cas systems have in common the genes for uptake of genetic information from the invading MGE but differ in the composition of the effector complex that directs and enforces the immune response. The genome of Mycobacterium tuberculosis encodes a Class 1, type III-A CRISPR-Cas system that has not been studied in detail.

Methodology

We used heterologous production of the mycobacterial CRISPR-Cas genes. Purification of the enzymes enabled in vitro biochemical characterisation of the adaptation, maturation and interference proteins. Reconstitution of the system in E. coli was used to demonstrate functionality in vivo.

Results

Maturation of crRNA through the action of Cas6 proceeded as expected, generating canonical CRISPR RNAs (crRNAs). The type III effector complex, consisting of Csm1-5, was shown to bind crRNA and cleave target RNA with the typical 6 nt spacing, display ssDNase activity and produce the cyclic oligoadenylate signalling molecule. The latter clearly activated the ribonuclease Csm6, an essential element in type III immunity. The M. tuberculosis type III CRISPR-Cas system was also reconstituted in E. coli where it provided plasmid immunity, demonstrating the functionality in vivo.

Conclusions

All elements of the M. tuberculosis type III CRISPR-Cas system are functional in vitro and in vivo. These studies lay the foundation for further investigations into the mechanism of adaptive immunity and possible applications in biotechnology.

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/content/journal/acmi/10.1099/acmi.ac2019.po0344
2019-03-01
2020-01-21
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http://instance.metastore.ingenta.com/content/journal/acmi/10.1099/acmi.ac2019.po0344
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