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Presentation of the laboratory and its research topics:
David Bikard is a new recruit of the Institut Pasteur and will start his lab in April 2014 within the Microbiology department as a member of the “Integrative Biology of Emerging Infectious Diseases” laboratory of excellence. The lab will focus on the many unresolved aspects of CRISPR biology and their application as tools to study and fight pathogenic bacteria.
Description of the project:
CRISPR loci, and their associated cas (CRISPR associated) genes, encode a sequence-specific defense mechanism against bacteriophages and plasmids . They consist in arrays of short repeats separated by “spacer” sequences captured from invading plasmids and phages. The CRISPR array is transcribed into small crRNA that can direct the Cas effector proteins to destroy target DNA. The discovery of RNA-guided nucleases among Cas proteins is a major finding that has led to the development of many tools for the purpose of genome editing and the control of gene expression [2-4]. The well-studied Cas9 protein has emerged as a universal and easy to use tool for biotechnological applications. A catalytically inactive mutant of Cas9 termed dCas9 has notably been used to repress gene expression [5, 6]. The dCas9 protein is able to specifically bind target DNA without cutting it, and efficiently blocks transcription.
The goal of this project is to develop dCas9 technology in Staphyloccus aureus, and use it to decipher virulence pathways. Staphyloccus aureus is a major source of respiratory tract and skin disease as well as a common cause of nosocomial infections. One of the major advantages of CRISPR based gene repression is that it is very easy to reprogram, and is thus amenable to high throughput screens. A library of crRNA will be constructed that will target every single gene in S. aureus genome. Another library will also be constructed that will target every pair of genes using a CRISPR arrays with two spacers. The fitness of individual clones in the libraries will be assessed in different conditions, including the infection of a mouse model. Fitness measurements can be achieved through deep-sequencing of the CRISPR array in the whole bacterial population by comparing the abundance of reads for each CRISPR locus before and after the experiment. The data recovered will be used to reconstruct gene networks and study virulence pathways.
This kind of high throughput approach is only becoming possible now, thanks to the emergence of new sequencing technologies as well as new CRISPR based tools. The large amount of data recovered will provide an unprecedented look at virulence pathways at the system level. We hope that this project will lead the path towards a broad adoption of this approach for the study of gene networks.
1. Wiedenheft B, Sternberg SH, Doudna JA: RNA-guided genetic silencing systems in bacteria and archaea. Nature 2012, 482(7385):331-338.
3. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA et al: Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 2013, 339(6121):819-823.
4. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R: One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell 2013, 153(4):910-918.
5. Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA: Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res 2013.
6. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA: Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013, 152(5):1173-1183.
CRISPR, Gene Regulation, Systems Biology, Virulence
Expected profile of the candidate (optional):
A curious, agile mind and a good work ethic are what matter most. The rest can be learned. With this in mind, some experience in molecular biology or microbiology is appreciated. Some computational skills might also be a plus for the data analysis.