Saving Bacteria from Diseases and the Cas9-CRISPR Revolution
Most NIH-backed funding proposals discuss their plans to wipe out pathogens like Streptococcus from the face of earth, but some other souls do like those bacteria and care for their well-being. Fortunately, one such group did not need government to pay for their research and figured out how to save Streptococcus from their own diseases. The methods derived by them have become a major part of new genetic revolution.
We should mention at the outset that although some Streptococcus strains can lead to diseases ranging from pink eye, pneunomonia, meningitis to ‘flesh- eating’ bacterial infections, other ones are harmless or often friendly. Especially _Streptococcus thermophilus_ is widely used in the dairy industry in cheese and yogurt production.
P. Horvath and his colleagues at Danisco noticed that domesticated strains of Streptococcus were generally prone to phage infection, but some phage- resistant strains had extra nucleotide sequences in the genome, possibly to protect them from infection. Those nucleotide sequences appeared in a strange part of the genome - within short repeat sequences. In fact, those repeats were not straightforward repeats, but repetitive sequences separated by short (~24-48 nt) spacer sequences. Based on extensive experimentation, they established that those repeat-spacer arrays were part of an ‘adaptive immune system’ to protect the bacteria from phage infection (CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes). A possible mechanism is shown below -
Since then, the cas-CRISPR system has been adapted to make an extremely versatile method for DNA editing. We will cover a number of other commentaries on related papers, but for the time being, please check the following papers -
2. Review paper by Jennifer Doudna (get pdf here)
RNA-guided genetic silencing systems in bacteria and archaea
3. Cracking CRISPR - a 2009 interview of Horvath
4. RNA-guided human genome engineering via Cas9 - George Church paper
5. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.
6. Multiplex Genome Engineering Using CRISPR/Cas Systems
