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Team:Arizona State/Project
From 2011.igem.org
Project
Contents |
Overall project
- Our project will have several stages, all pursuant to the general investigation and modularization of the CRISPR pathway:
- Proof of concept targeting reporters such as GFP, eventually creating a CRISPR biobrick
- Investigate CRISPR system dynamics based on factors such as degradation of self-targeting sequences and maintenance of the array.
- Target genes such as NDM-1 or other clinically relevant pathways.
NDM-1 in Perspective
Global antibiotic resistance is a concern of the utmost importance to the World Health Organization and health care everywhere. Bacteria that have acquired antibiotic resistance jeopardize world health care as a whole, because they increase mortality rate of normally curable infections, and there is no coherent approach to containing and countering resistant strains. New Delhi Metallo‐Beta‐Lactamse (NDM‐1) containing bacteria are particularly ominous because the NDM‐1 enzyme hydrolyzes a broad range of potent beta‐lactam antibiotics (e.g. carbapenems). This enzyme is effective in rendering normal lines of treatment for bacterial infection useless. NDM‐1 positive strains originated in India and Pakistan and have recently spread to the UK, Europe, and Canada. There has also been a drastic increase in the number of reported NDM‐1 positive cases in the United States, according to the Centers of Disease Control and Prevention. Viable antibiotics as a resource are becoming more and more deficient. Alternative solutions to resistance must be promptly sought and intelligently employed to counter the threat of antibiotic resistant bacteria.
The CRISPR Mechanism
The CRISPR‐Cas pathway can be compared to a prokaryotic immunity or RNA interference that can be directed to silence a gene of interest. This mechanism of bacterial survival affords us an interesting method to tackle the aforementioned problem. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene loci have been demonstrated to equip both prokaryotes and archaea with a defense mechanism against exogenous DNA and RNA sequences. 1,2 CRISPR genes appear in an array that contains contiguous spacers, repeats, and an operon of structural genes. The transcripts from the spacer/repeat region undergo hair pinning due to the palindromic sequence structure. The peptide products of the CRISPR‐associated structural genes (CAS) work cooperatively with crRNA to silence a complimentary target (Diagram 1). 3 The function is a prokaryotic analog to both RNA interference and immunity. CRISPR quickly presents itself as a potentially useful tool in prokaryotic gene manipulation. Our goal as ASU’s first iGEM team is to develop a CRISPR plasmid that contains elements to target and silence the NDM‐1 gene sequence (Diagram 2). While targeting NDM‐1, we recognize that CRISPR can potentially target any gene of interest, thus we will develop a robust platform for gene silencing. The final product of this project will be a fully functioning CRISPR array that will be submitted to the Standard Registry of Biological Parts, an open‐source collection of DNA building blocks, as a BioBrick, a modular component for genetic engineering (Diagram 3).
CRISPR / CAS / RAMP Overview
References
- Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero D, Horvath P. CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science 315, 1709‐1712 (2007).
- Karginov FV, Hannon GJ. The CRISPR system: small RNA‐guided defense in bacteria and archaea. Molecular Cell 37, 7‐19 (2010).
- Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960‐964 (2008).
