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From 2013.igem.org

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CRISPR

Molecular biology has long exploited phage machinery as tools for manipulating genomes. Recently, the literature has exploded with work on the CRISPR adaptive immune system of prokaryotes, which involves nucleases that can be guided by small RNAs. The system includes loci composed of a series of repeats separated by ‘spacer’ elements that match a target sequence. This region is transcribed into the small RNAs that specify the target sequence (or protospacer) to be cleaved by a CRISPR-associated gene, Cas9. Cas 9 is a double stranded DNA endonuclease with single nucleotide resolution. Several groups have demonstrated that the CRISPR system is extremely useful for genome editing, which inspired us to re-factor it for our own purposes.

As an adaptive immune response, we wanted to know if CRISPR could be put together in a modular way to confer resistance to known phage genomes - vaccinating the host. We first wrote programs capable of predicting the most broadly neutralizing spacer region from a file of compiled phage genomes. We then assembled the minimum components biobricks to conduct the necessary proof-of-concept experiments in E.coli. Ultimately, we hope that large-scale fermenters could be vaccinated against collapse caused by environmental phage infection. To extend the application of this approach, we designed specifically neutralization elements that allow for population level programming of a culture. We envision this being first applied to yogurt where, for example, the biosynthesis of flavor combinations is controlled by targeted phage addition.

Spacer Selection