Anti-CRISPRs (Acr) are proteins that are made by bacteriophages (viruses that infect bacteria) to suppress the CRISPR-Cas defence system in bacteria. Due to this, bacteriophages can avoid being destroyed by bacteria and persist to invade them. There are many acr that have been discovered, and these can be categorised depending on which specific component of the CRISPR-Cas system they inhibit. Interestingly, the genes encoding acr can be found in the tail, capsid, or the extreme end of phages. AcrF1, AcrF2, AcrF3, AcrF4 and AcrF5 were the first acr families discovered in phages that infect Pseudomonas aeruginosas.
So how do Acr work?
There are 3 main methods in which Acr can inhibit Cas activity:
- Blocking the ability of Cas to cleave the DNA.
- Blocking PAM recognition.
- Inhibition of the gRNA-Cas complex.
AcrIIA2 and AcrIIA4 are notable anti-CRISPR proteins that inhibit the activity of Cas9 by preventing DNA binding. AcrIIA4 prevents Cas9 from recognising the PAM on the target sequence and thus prevents the CRISPR-Cas complex binding to the target DNA. Furthermore, there is AcrIIC1 which inhibits CRISPR-Cas9 activity too but using a different method. AcrIIC1 binds to the HNH domain on Cas9, and this prevents Cas9 cleaving the target DNA. These are just examples using Cas9, there are several acr that can inhibit the other Cas variants such as Cas12 and Cas3
Anti-CRISPRs could be used for many purposes. Firstly, acr could be used to detect the amount of Cas and its activity from samples. Phaneuf and colleagues were able to detect the levels of Cas9 by using AcrIIC1 as a capture reagent. This is helpful when wanting to determine which specific Cas is present.
Gene drives have been developed to promote specific traits within certain populations such as engineering mosquitoes to prevent the spread of malaria or dengue. Researchers suggest that gene drives should be used with caution as sometimes the technology may not work as expected. Therefore, control measures should be adopted in case of undesirable effects, Acr could be used to modulate these gene drives. AcrIIA4 was able to inhibit a gene drive model in yeast in Basgall's study.
Acr could also be used to decrease the off-target effects associated with CRISPR-Cas editing. In a study by Shin, it was found that adding a small amount of AcrIIA4 after CRISPR gene editing, reduced the off-target effects of Cas9. There is a biotech company that is already conducting research into this. Acrigen is based in the US and was co-founded by Drs David Rabuka and Joe Bondy-Denomy. Acrigen aims to make CRISPR editing safer using anti-CRISPRs.
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