Anti-CRISPR

Anti-CRISPR (Anti-Clustered Regularly Interspaced Short Palindromic Repeats or Acr) is a group of proteins found in phages, that inhibit the normal activity of CRISPR-Cas, the immune system of certain bacteria. CRISPR consists of genomic sequences that can be found in prokaryotic organisms, that come from bacteriophages that infected the bacteria beforehand, and are used to defend the cell from further viral attacks. Anti-CRISPR results from an evolutionary process occurred in phages in order to avoid having their genomes destroyed by the prokaryotic cells that they will infect.

Before the discovery of this type of family proteins, the acquisition of mutations was the only way known that phages could use to avoid CRISPR-Cas mediated shattering, by reducing the binding affinity of the phage and CRISPR. Nonetheless, bacteria have mechanisms to retarget the mutant bacteriophage, a process that it is called "priming adaptation". So, as far as researchers currently know, anti-CRISPR is the most effective way to ensure the survival of phages throughout the infection process of bacteria.

History

Anti-CRISPR systems were first seen in '' Pseudomonas aeruginosa'' prophages, which disabled type I-F CRISPR–Cas system, characteristic of some strains of these bacteria. After analysing the genomic sequences of these phages, genes codifying five different Anti-CRISPR proteins (also named Acrs) were discovered. Such proteins were AcrF1, AcrF2, AcrF3, AcrF4 and AcrF5. Research found none of these proteins disrupted the expression of Cas genes nor the assembling of CRISPR molecules, so it was thought that those type I-F proteins directly affected the CRISPR–Cas interference.

Further investigation confirmed this hypothesis with the discovery of 4 other proteins (AcrE1, AcrE2, AcrE3 and AcrE4), which were shown to impede ''Pseudomonas aeruginosa''’s CRISPR-Cas system. Furthermore, the locus of the genes codifying these type I-E proteins was really close to the one responsible for the type I-F proteins expression in the same group of phages, leading to the conclusion that both types of proteins worked together. However, these first nine proteins shared no common sequence motifs, which would have made easier the identification of new Anti-CRISPR protein families.

Later on, it was seen that phages that produced such proteins also encoded a putative transcriptional regulator named Aca 1 (anti-CRISPR associated 1) which was genetically located really close to the anti-CRISPR genes. This regulatory protein is supposed to be the responsible for the anti-CRISPR gene expression during the infectious cycle of the phage, therefore, both types of proteins (anti-CRISPR and Aca1) seem to work together as a single mechanism.

After some studies, a similar amino-acid sequence to that of Aca1 was found, leading to the discovery of Aca2, a new family of Aca proteins. Aca2 also revealed the existence of five new groups of type I-F anti-CRISPR proteins due to their genomic proximity: AcrF6, AcrF7, AcrF8, AcrF9 and AcrF10. These proteins were not only present in ''Pseudomonas aeruginosa''’s phages, as they also affected other cells of the ''Pseudomonadota'' (formerly ''Proteobacteria'').

Thanks to the use of bioinformatic tools, in 2016, AcrIIC1, AcrIIC2 and AcrIIC3 protein families were discovered in '' Neisseria meningitidis'' (which had been infected by phages previously). Such proteins were the first inhibitors of type II CRISPR–Cas to be found (concretely, they impeded II-C CRISPR–Cas9, the type of mechanism used in the genetic edition of human cells). A year later, a study confirmed the presence of type II-A CRISPR–Cas9 inhibitors (AcrIIA1, AcrIIA2, AcrIIA3 and AcrIIA4) in '' Listeria monocytogenes'' (infected by bacteriophages which introduced the anti-CRISPR proteins). Two of those proteins (AcrIIA2 and AcrIIA4) were demonstrated to work properly against '' Streptococcus pyogenes'' type II-A defensive CRISPR system.

The result of all this research has been the discovery of 21 different Anti-CRISPR protein families, despite other inhibitors may exist due to the quick mutational process of phages. Thus, more research is needed to unravel the complexity of anti-CRISPR systems.

Types

Anti-CRISPR genes can be found in different parts of the phage DNA: in the capsid, the tail and at the extreme end. Moreover, it has been found that many MGEs have two or even three Acr genes in a single operon, which suggest that they could have been exchanged between MGEs.

As all proteins, Acr family proteins are formed by the translation and transduction of the genes, and their classification is based on the type of CRISPR-Cas system they inhibit, due to the fact that each anti-CRISPR protein inhibits a specific CRISPR-Cas system. Although not many anti-CRISPR proteins have been discovered, these are the ones that have been found so far:

So far, genes encoding anti-CRISPR proteins have been found in myophages, siphophages, putative conjugative elements