Anti-CRISPR

{{Short description|Group of proteins found in phages}} {{Infobox nonhuman protein | Name = Anti-CRISPR (AcrIIA4 protein) | Symbol = AcrIIA4 | UniProt = A0A247D711 | Organism = Listeria monocytogenes prophages | image = AcrIIA4 structure PDB.jpg | caption = AcrIIA4 structure obtained from PDB with JSmol viewer. | PDB = 5XN4 | GenLoc_end = | width = | TaxID = }} {{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.<ref name = "Nakamura_2019">{{cite journal | vauthors = Nakamura M, Srinivasan P, Chavez M, Carter MA, Dominguez AA, La Russa M, Lau MB, Abbott TR, Xu X, Zhao D, Gao Y, Kipniss NH, Smolke CD, Bondy-Denomy J, Qi LS | display-authors = 6 | title = Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells | journal = Nature Communications | volume = 10 | issue = 1 | page = 194 | date = January 2019 | pmid = 30643127 | doi = 10.1038/s41467-018-08158-x | pmc = 6331597 | bibcode = 2019NatCo..10..194N }}</ref> 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.<ref>{{cite journal | vauthors = Barrangou R | title = The roles of CRISPR-Cas systems in adaptive immunity and beyond | journal = Current Opinion in Immunology | volume = 32 | pages = 36–41 | date = February 2015 | pmid = 25574773 | doi = 10.1016/j.coi.2014.12.008 }}</ref> 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.<ref>{{cite journal | vauthors = Stanley SY, Borges AL, Chen KH, Swaney DL, Krogan NJ, Bondy-Denomy J, Davidson AR | title = Anti-CRISPR-Associated Proteins Are Crucial Repressors of Anti-CRISPR Transcription | journal = Cell | volume = 178 | issue = 6 | pages = 1452–1464.e13 | date = September 2019 | pmid = 31474367 | doi = 10.1016/j.cell.2019.07.046 | pmc = 6754177 }}</ref>

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.<ref>{{cite journal | vauthors = Maxwell KL | title = The Anti-CRISPR Story: A Battle for Survival | journal = Molecular Cell | volume = 68 | issue = 1 | pages = 8–14 | date = October 2017 | pmid = 28985512 | doi = 10.1016/j.molcel.2017.09.002 | doi-access = free }}</ref>

== History == Anti-CRISPR systems were first seen in ''Pseudomonas aeruginosa'' prophages,<ref name="Bondy-Denomy_2013">{{cite journal | vauthors = Bondy-Denomy J, Pawluk A, Maxwell KL, Davidson AR | title = Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system | journal = Nature | volume = 493 | issue = 7432 | pages = 429–32 | date = January 2013 | pmid = 23242138 | doi = 10.1038/nature11723 | pmc = 4931913 | bibcode = 2013Natur.493..429B }}</ref> 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.<ref name="Pawluk_2018">{{cite journal | vauthors = Pawluk A, Davidson AR, Maxwell KL | title = Anti-CRISPR: discovery, mechanism and function | journal = Nature Reviews. Microbiology | volume = 16 | issue = 1 | pages = 12–17 | date = January 2018 | pmid = 29062071 | doi = 10.1038/nrmicro.2017.120 | s2cid = 13222384 }}</ref>

Further investigation confirmed this hypothesis with the discovery of 4 other proteins ('''AcrE1''', '''AcrE2''', '''AcrE3''' and '''AcrE4'''), which were shown to impede ''Pseudomonas aeruginosa''<nowiki />'s CRISPR-Cas system.<ref name = "Pawluk_2014">{{cite journal | vauthors = Pawluk A, Bondy-Denomy J, Cheung VH, Maxwell KL, Davidson AR | title = A new group of phage anti-CRISPR genes inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa | journal = mBio | volume = 5 | issue = 2 | pages = e00896 | date = April 2014 | pmid = 24736222 | pmc = 3993853 | doi = 10.1128/mBio.00896-14 | doi-access = free}}</ref> 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.<ref>{{cite journal | vauthors = Borges AL, Davidson AR, Bondy-Denomy J | title = The Discovery, Mechanisms, and Evolutionary Impact of Anti-CRISPRs | journal = Annual Review of Virology | volume = 4 | issue = 1 | pages = 37–59 | date = September 2017 | pmid = 28749735 | pmc = 6039114 | doi = 10.1146/annurev-virology-101416-041616 }}</ref> 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 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.<ref name="Bondy-Denomy_2013"/>

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''<nowiki />'s phages, as they also affected other cells of the ''Pseudomonadota'' (formerly ''Proteobacteria'').<ref name="Pawluk_2018" />

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).<ref name="Pawluk 1829–1838.e9">{{cite journal | vauthors = Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR | display-authors = 6 | title = Naturally Occurring Off-Switches for CRISPR-Cas9 | journal = Cell | volume = 167 | issue = 7 | pages = 1829–1838.e9 | date = December 2016 | pmid = 27984730 | pmc = 5757841 | doi = 10.1016/j.cell.2016.11.017 }}</ref> 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.

While the mechanistical bases for many Acrs against other CRISPR systems were well known, only few anti-CRISPRs against the complex III-A CRISPR system had been well characterised before the finding of '''AcrIIIA2''' encoded by ''Streptococcus thermophilus'' phage SW3. With the use of bioinformatic tools, phages infecting ''Streptococcus'' species were screened with a quilt-by-association method. Adjacent to the gene encoding the previously known anti-CRISPR protein '''AcrIIA6''' against type II-A CRIPSR systems an anti-CRISPR protein AcrIIIA2 against type III-A-Cas was discovered. It blocks the seed region of crRNA and thereby the binding of target RNA by forming a complex with the host bacterium’s enolase enzyme. This blocks all downstream functions of the type III CRISPR: cleavage of target RNA, cleavage of DNA, and cyclic oligoadenylate synthesis.<ref>{{Cite journal |last=Johnson |first=Katie A. |last2=Goswami |first2=Hemant N. |last3=Catchpole |first3=Ryan J. |last4=Ahmadizadeh |first4=Fozieh |last5=Zhao |first5=Peng |last6=Wells |first6=Lance |last7=Li |first7=Hong |last8=Terns |first8=Michael P. |date=December 2025 |title=A phage-encoded anti-CRISPR protein co-opts host enolase to prevent type III CRISPR immunity |url=https://www.nature.com/articles/s41564-025-02178-2 |journal=Nature Microbiology |language=en |volume=10 |issue=12 |pages=3162–3175 |doi=10.1038/s41564-025-02178-2 |issn=2058-5276 |pmc=12811900 |pmid=41219509}}</ref>

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.<ref name="Pawluk_2016" />

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: {| class="wikitable" |+ Anti-CRISPR protein families (''table adapted from a reference'')<ref name="Pawluk_2018" /> !Anti-CRISPR protein family !Characterized member !CRISPR system inhibited !Number of amino acids |- |AcrE1 |JBD5‑34 (''Pseudomonas aeruginosa'') |I‑E |100 |- |AcrE2 |JBD88a‑32 (''P. aeruginosa'') |I‑E |84 |- |AcrE3 |DMS3‑30 (''P. aeruginosa'') |I‑E |68 |- |AcrE4 |D3112‑31 (''P. aeruginosa'') |I‑E |52 |- |AcrF1 |JBD30‑35 (''P. aeruginosa'') |I‑F |78 |- |AcrF2 |D3112‑30 (''P. aeruginosa'') |I‑F |90 |- |AcrF3 |JBD5‑35 (''P. aeruginosa'') |I‑F |139 |- |AcrF4 |JBD26‑37 (''P. aeruginosa'') |I‑F |100 |- |AcrF5 |JBD5‑36 (''P. aeruginosa'') |I‑F |79 |- |AcrF6 |AcrF6''Pae'' (''P. aeruginosa'') |I‑E and I‑F |100 |- |AcrF7 |AcrF7''Pae'' (''P. aeruginosa'') |I‑F |67 |- |AcrF8 |AcrF8''ZF40'' (''Pectobacterium'' phage ZF40) |I‑F |92 |- |AcrF9 |AcrF9''Vpa'' (''Vibrio parahaemolyticus'') |I‑F |68 |- |AcrF10 |AcrF10''Sxi'' (''Shewanella xiamenensis'') |I‑F |97 |- |AcrIIA1 |AcrIIA1''Lmo'' (''Listeria monocytogenes'') |II‑A |149 |- |AcrIIA2 |AcrIIA2''Lmo'' (''L. monocytogenes'') |II‑A |123 |- |AcrIIA3 |AcrIIA3''Lmo'' (''L. monocytogenes'') |II‑A |125 |- |AcrIIA4 |AcrIIA4''Lmo'' (''L. monocytogenes'') |II‑A |87 |- |AcrIIC1 |AcrIIC1''Nme'' (''Neisseria meningitidis'') |II‑C |85 |- |AcrIIC2 |AcrIIC2''Nme'' (''N. meningitidis'') |II‑C |123 |- |AcrIIC3 |AcrIIC3''Nme'' (''N. meningitidis'') |II‑C |116 |}

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

Attempts have been made to find common surrounding genetic features of anti-CRISPR genes, but without any success. Nevertheless, the presence of an ''aca'' gene just below anti-CRISPR genes has been observed.<ref name="Pawluk_2016" />

The first Acr protein families to be discovered were AcrF1, AcrF2, AcrF3, AcrF4 and AcrF5.<ref name="Bondy-Denomy_2013" /> These inhibitors are mainly found in ''Pseudomonas'' phages, which are capable of infecting ''Pseudomonas aeruginosas'' possessing a type I‑F CRISPR–Cas system. Then, in another study, AcrE1, AcrE2, AcrE3 and AcrE4 protein families were found to also inhibit the type I‑F CRISPR–Cas in ''Pseudomonas aeruginosas.''<ref name="Pawluk_2014" />

Later on, AcrF6, AcrF7, AcrF8, AcrF9 and AcrF10 protein families, which were also able to inhibit type I‑F CRISPR–Cas, were found to be very common in Pseudomonadota MGEs.<ref name="Pawluk_2016">{{cite journal | vauthors = Pawluk A, Staals RH, Taylor C, Watson BN, Saha S, Fineran PC, Maxwell KL, Davidson AR | display-authors = 6 | title = Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species | journal = Nature Microbiology | volume = 1 | issue = 8 | page = 16085 | date = June 2016 | pmid = 27573108 | doi = 10.1038/nmicrobiol.2016.85 | s2cid = 3826582 }}</ref>

The first inhibitors of a type II CRISPR–Cas system were then discovered: AcrIIC1, AcrIIC2 and AcrIIC3, that block the type II‑C CRISPR–Cas9 activity of ''Neisseria meningitidis.''<ref name="Pawluk 1829–1838.e9" />

Finally, AcrIIA1, AcrIIA2, AcrIIA3 and AcrIIA4 were found. These protein families have the ability to inhibit the type II‑A CRISPR–Cas system of ''Listeria monocytogenes.''<ref>{{cite journal | vauthors = Rauch BJ, Silvis MR, Hultquist JF, Waters CS, McGregor MJ, Krogan NJ, Bondy-Denomy J | title = Inhibition of CRISPR-Cas9 with Bacteriophage Proteins | journal = Cell | volume = 168 | issue = 1–2 | pages = 150–158.e10 | date = January 2017 | pmid = 28041849 | pmc = 5235966 | doi = 10.1016/j.cell.2016.12.009 }}</ref>

As for the naming convention of Acr family proteins, it is established as follows: firstly, the type of system inhibited, then a numerical value referring to the protein family and finally the source of the specific anti-CRISPR protein. For example, AcrF9''_Vpa'' is active against the type I-F CRISPR–Cas system. It also was the ninth anti-CRISPR described for this system, and it is encoded in an integrated MGE in a ''Vibrio parahaemolyticus'' genome.

== Structure == As exposed above, there is a wide spectrum of anti-CRISPR proteins, but few of these have been deeply studied. One of the most studied and well-defined Acrs is AcrIIA4, which inhibits Cas9, thus blocking the II-A CRISPR-Cas system of ''Streptococcus pyogenes''.

=== AcrIIA4 === [[File:AcrIIA4.jpg|thumb|frameless

Structure of AcrIIA4 obtained with the UCSF Chimera software,<ref>{{cite web |url= https://www.cgl.ucsf.edu/chimera/|title=UCSF Chimera |website=Chimera|access-date=25 October 2019 }}</ref> where its PDB file was uploaded.<ref name="PDB_5XN4" /> Different colours were assigned to the four different secondary structures found in this protein: blue for β-strands, red for α-helices, orange for the 3₁₀ helix, and grey for loops. Originally, the PDB file contains the 20 lowest energy sequences (and thus, the most stable ones) superposed, one of which was randomly selected to create the figure.<ref name="Kim_2018" />]] The protein was solved using nuclear magnetic resonance (NMR); it contains 87 residues and its molecular weight is 10.182 kDa.<ref name="PDB_5XN4">{{cite journal |url= http://www.rcsb.org/structure/5XN4 |title=AcrIIA4 - PDB |website=Protein Data Bank|access-date=2019-10-15|doi=10.2210/pdb5xn4/pdb |url-access=subscription }}</ref> AcrIIA4 contains:

* '''3 antiparallel β-strands''' (the first, from residues 16 to 19, the second, from 29 to 33, and the third, from 40 to 44) that form a β-sheet. This represents a 16,1% of the total number of amino acids, as 14 of them form the β-strands. * '''3 α-helices''' (the first, 2–13 residues, the second, 50–59 residues, and the third, 68–85 residues). * '''1 3₁₀ helix''' placed between the first (β1) and second (β2) β-strands, which starts at residue 22 and end in residue 25. The total helical part is composed of 40 residues, which is a 50,6% of the protein. * '''Loops''' joining the different secondary structures.

There is a good definition of the secondary structures, as the three α-helices are packed near the three β-strands. Strikingly, between β3 strand, α2 and α3 helices, there is a hydrophobic core, originated by a cluster of aromatic side chains which are attracted by non-covalent interactions, such as pi stacking. Moreover, as it is an acidic protein, there is a high concentration of negatively charged residues in the loops between β3 and α2, between α2 and α3, and in the first part of α3, which may play an important role in the inhibition of Cas9, as negative charges might imitate phosphates of nucleic acids.<ref name="Kim_2018">{{cite journal | vauthors = Kim I, Jeong M, Ka D, Han M, Kim NK, Bae E, Suh JY | title = Solution structure and dynamics of anti-CRISPR AcrIIA4, the Cas9 inhibitor | journal = Scientific Reports | volume = 8 | issue = 1 | page = 3883 | date = March 2018 | pmid = 29497118 | pmc = 5832863 | doi = 10.1038/s41598-018-22177-0 | bibcode = 2018NatSR...8.3883K }}</ref>

=== AcrF1 === On the other hand, there is another Acr, AcrF1, which may not have been as studied as the explained above, although there is a good description of its structure. It inhibits the I-F CRISPR-Cas system of ''Pseudomonas aeruginosa''. Maxwell et al.<ref name="Maxwell_2016">{{cite journal | vauthors = Maxwell KL, Garcia B, Bondy-Denomy J, Bona D, Hidalgo-Reyes Y, Davidson AR | title = The solution structure of an anti-CRISPR protein | journal = Nature Communications | volume = 7 | issue = 1 | article-number = 13134 | date = October 2016 | pmid = 27725669 | pmc = 5062604 | doi = 10.1038/ncomms13134 | bibcode = 2016NatCo...713134M }}</ref> solved the 3D structure using NMR.

The protein contains 78 residues,<ref name="Pawluk_2018" /> between which interact to form secondary structures. The structure of AcrF1 is formed of two anti-parallel α-helices and a β-sheet, which contains four anti-parallel β-strands. This β-sheet is placed in the contrary side of the α-helical part, which creates a hydrophobic core formed of 13 amino acids. Turns can also be found in different parts of the protein, for instance, joining the β-strands.<ref name="Maxwell_2016" /><ref>{{Cite journal|url=https://www.rcsb.org/structure/2LW5|title=AcrF1 - PDB|journal=Worldwide Protein Data Bank|access-date=2019-10-14|doi=10.2210/pdb2lw5/pdb| vauthors = Davidson AR, Pawluk A, Maxwell KL, Bondy-Denomy J |date=2014 |url-access=subscription}}</ref>

There are surface residues which actively participate in the active site of AcrF1, two of which are tyrosines (Y6 and Y20) and the third amino acid is a glutamic acid (E31), as their mutation by an alanine causes a 100-fold decrease in the activity of the protein (with Y20A and E31A mutations), and a 10⁷-fold decrease when Y6 is mutated.

The different structures that form the protein create a strange combination, as Maxwell et al. conducted a DALI search in order to find similarities between other proteins, and they found no informative similarities.<ref name="Maxwell_2016" />

== Function == === Avoiding destruction of the phage DNA === The principal function of anti-CRISPR proteins is to interact with specific components of CRISPR-Cas systems, such as the effector nucleases, to avoid the destruction of the phage DNA (by binding or cleavage).<ref name="van_Gent_2018">{{cite journal | vauthors = van Gent M, Gack MU | title = Viral Anti-CRISPR Tactics: No Success without Sacrifice | language = en | journal = Immunity | volume = 49 | issue = 3 | pages = 391–393 | date = September 2018 | pmid = 30231980 | doi = 10.1016/j.immuni.2018.08.023 | doi-access = free }}</ref>

A phage introduces its DNA into a prokaryotic cell, usually the cell detects a sequence known as "target", that activates CRISPR-Cas immune system, but the presence of an initial sequence (before the target) encoding the formation of Acr proteins, avoids phage destruction. Acr proteins are formed before the target sequence is read. This way, the CRISPR-Cas system is blocked before it can develop a response.

The procedure starts with the CRISPR locus being transcribed into crRNAs (CRISPR RNA). CrRNAs combine with Cas proteins forming a ribonucleoprotein complex called Cascade. This complex surveys the cell to find complementary sequences of the crRNA. When this sequence is found, the Cas3 nuclease is recruited to the Cascade, and the target DNA from the phage is cleaved. But, for instance, when AcrF1 and AcrF2 are found (anti-CRISPR proteins), these interact with Cas7f and Cas8f-Cas5f, respectively, not allowing the binding to the phage DNA. Moreover, the cleaving of the target is prevented by the union between AcrF3 and Cas3.<ref name="Pawluk_2018" />

[[File:Phage cooperation against CRISPR immunity.png|thumb|290x290px|'''Phage-phage cooperation:''' First phage infections may be unable to hamper the CRISPR immunity, but phage-phage cooperations increasingly boost Acr production and host immunosuppression, which produces an increase on the vulnerability of the host cell to reinfection, and finally allows a successful infection and spreading of a second phage.

''<small>Based on a representation found in the 17th reference.</small>'' <ref name="van_Gent_2018" />]]

The majority of Acr genes are located next to anti-CRISPR-associated (Aca) genes, which encode proteins with a helix-turn-helix DNA-binding motif. Aca genes are preserved, and researchers are using them to identify Acr genes, but the function of the proteins they encode is not totally clear. The Acr-associated promoter produces high levels of Acr transcription just after the phage DNA injection into the bacteria takes place and, afterward, Aca proteins repress the transcription. If this wasn't repressed, the constant transcription of the gene would be lethal to the phage. Therefore, Aca activity is essential to ensure its survival.<ref>{{Cite web|url=https://sso.ub.edu/CAS/index.php/login?service=https%3a%2f%2flogin.sire.ub.edu%2flogin%3fqurl%3dezp.2aHR0cHM6Ly93d3cuY2VsbC5jb20vY2VsbC9mdWxsdGV4dC9TMDA5Mi04Njc0KDE5KTMwODQ2LTM.X3JldHVyblVSTD1odHRwczovL2xpbmtpbmdodWIuZWxzZXZpZXIuY29tL3JldHJpZXZlL3BpaS9TMDA5Mjg2NzQxOTMwODQ2Mz9zaG93YWxsPXRydWU-|title=Inici sessió - Identificació UB - Universitat de Barcelona|website=sso.ub.edu|access-date=2019-10-25}}</ref>

=== Phage-phage cooperation === Moreover, it has been verified that bacteria with CRISPR-Cas systems are still partially immune to Acr. Consequently, initial abortive phage infections may be unable to hamper CRISPR immunity, but phage-phage cooperation can increasingly boost Acr production and promote immunosuppression, which might produce an increase on the vulnerability of the host cell to reinfection, and finally allow a successful infection and spreading of a second phage.<ref name="van_Gent_2018" /> This cooperation creates an epidemiological tipping point, in which, depending on the initial density of Acr-phages and the strength of CRISPR/Acr binding, phages can either be eliminated or originate a phage epidemic (the number of bacteriophages is amplified).<ref name="Landsberger_2018">{{cite journal | vauthors = Landsberger M, Gandon S, Meaden S, Rollie C, Chevallereau A, Chabas H, Buckling A, Westra ER, van Houte S | display-authors = 6 | title = Anti-CRISPR Phages Cooperate to Overcome CRISPR-Cas Immunity | journal = Cell | volume = 174 | issue = 4 | pages = 908–916.e12 | date = August 2018 | pmid = 30033365 | pmc = 6086933 | doi = 10.1016/j.cell.2018.05.058 }}</ref><ref name=":0">{{cite journal | vauthors = Borges AL, Zhang JY, Rollins MF, Osuna BA, Wiedenheft B, Bondy-Denomy J | title = Bacteriophage Cooperation Suppresses CRISPR-Cas3 and Cas9 Immunity | journal = Cell | volume = 174 | issue = 4 | pages = 917–925.e10 | date = August 2018 | pmid = 30033364 | pmc = 6086726 | doi = 10.1016/j.cell.2018.06.013 }}</ref>

If the starting levels of phages are high enough, the density of immunosuppressed hosts reaches a critical point where there are more successful infections than unsuccessful ones. Then, an epidemic begins. If this point is not reached, phage extinction occurs, and immunosuppressed hosts recover their initial state.<ref name="Landsberger_2018" /><ref name=":0" />

=== Phage immune evasion === It has become clear that Acr proteins play an important role in allowing phage immune evasion, though it is still unclear how anti-CRISPR proteins synthesis can overcome the host's CRISPR-Cas system, which can shatter the phage genome within minutes after the infection.<ref name="van_Gent_2018" />

== Mechanisms == [[File:Type I-F CRISPR-Cas system and inhibition mechanisms of three type I-F anti-CRISPRs..png|thumb|392x392px|Diagram showing type I-F CRISPR-Cas system, as well as inhibition mechanisms of three type I-F anti-CRISPRs. Type I-F CRISPR complex is made of 60 crRNA nucleotides and nine Cas proteins (the protein type is specified by the numbers 5,8,7,6). AcrF1 goes to Cas7f, preventing target DNA access to the crRNA guide. AcrF2 interacts both with Cas8f and Cas7f, difficulting target DNA access to the binding pocket. Finally, AcrF3 forms a homodimer, interacting with Cas3 by preventing its contact with the Cascade complex. ''Based on a representation from a review found in the references below.'' <ref>{{cite journal | vauthors = Zhu Y, Zhang F, Huang Z | title = Structural insights into the inactivation of CRISPR-Cas systems by diverse anti-CRISPR proteins | journal = BMC Biology | volume = 16 | issue = 1 | page = 32 | date = March 2018 | pmid = 29554913 | pmc = 5859409 | doi = 10.1186/s12915-018-0504-9 | doi-access = free }}</ref>|alt=]] Within all the Anti-CRISPR proteins that have been discovered so far, mechanisms have been described for only 15 of among them. These mechanisms can be divided into three different types: crRNA loading interference, DNA binding blockage and DNA cleavage prevention.

=== CrRNA loading interference === CrRNA (CRISPR RNA) loading interference mechanism has been mainly associated with the AcrIIC2 protein family.<ref name="Zhu_2019">{{cite journal | vauthors = Zhu Y, Gao A, Zhan Q, Wang Y, Feng H, Liu S, Gao G, Serganov A, Gao P | display-authors = 6 | title = Diverse Mechanisms of CRISPR-Cas9 Inhibition by Type IIC Anti-CRISPR Proteins | journal = Molecular Cell | volume = 74 | issue = 2 | pages = 296–309.e7 | date = April 2019 | pmid = 30850331 | pmc = 6750902 | doi = 10.1016/j.molcel.2019.01.038 }}</ref> In order to block Cas9 activity, it prevents the correct assembly of the crRNA-Cas9 complex.

=== DNA binding blockage === AcrIIC2 has been shown not to be the only one capable of blocking DNA binding. There are 11 other Acr family proteins that can also carry it out. Some among those are AcrIF1, AcrIF2, and AcrIF10, which act on different subunits of the Cascade effector complex of the type I-F CRISPR-Cas system, preventing the DNA to bind to the complex.<ref name="Bondy-Denomy_2015">{{cite journal | vauthors = Bondy-Denomy J, Garcia B, Strum S, Du M, Rollins MF, Hidalgo-Reyes Y, Wiedenheft B, Maxwell KL, Davidson AR | display-authors = 6 | title = Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins | journal = Nature | volume = 526 | issue = 7571 | pages = 136–9 | date = October 2015 | pmid = 26416740 | pmc = 4935067 | doi = 10.1038/nature15254 | bibcode = 2015Natur.526..136B }}</ref>

Furthermore, AcrIIC3 prevents DNA binding by promoting dimerization of Cas9 <ref name="Zhu_2019" /><ref name="Harrington_2017">{{cite journal | vauthors = Harrington LB, Doxzen KW, Ma E, Liu JJ, Knott GJ, Edraki A, Garcia B, Amrani N, Chen JS, Cofsky JC, Kranzusch PJ, Sontheimer EJ, Davidson AR, Maxwell KL, Doudna JA | display-authors = 6 | title = A Broad-Spectrum Inhibitor of CRISPR-Cas9 | journal = Cell | volume = 170 | issue = 6 | pages = 1224–1233.e15 | date = September 2017 | pmid = 28844692 | pmc = 5875921 | doi = 10.1016/j.cell.2017.07.037 }}</ref> and AcrIIA2 mimics DNA, thereby blocking the PAM recognition residues and consequently preventing dsDNA ''(double-stranded DNA)'' recognition and binding.<ref name = "Shin_2017">{{cite journal | vauthors = Shin J, Jiang F, Liu JJ, Bray NL, Rauch BJ, Baik SH, Nogales E, Bondy-Denomy J, Corn JE, Doudna JA | display-authors = 6 | title = Disabling Cas9 by an anti-CRISPR DNA mimic | journal = Science Advances | volume = 3 | issue = 7 | article-number = e1701620 | date = July 2017 | pmid = 28706995 | pmc = 5507636 | doi = 10.1126/sciadv.1701620 | bibcode = 2017SciA....3E1620S }}</ref><ref>{{cite journal | vauthors = Guo M, Wang S, Zhu Y, Wang S, Xiong Z, Yang J, Xu Z, Huang Z | display-authors = 6 | title = Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein | journal = Nature | volume = 546 | issue = 7658 | pages = 436–439 | date = June 2017 | pmid = 28448066 | doi = 10.1038/nature22377 | bibcode = 2017Natur.546..436D | s2cid = 4445217 }}</ref>

=== DNA cleavage prevention === AcrE1, AcrIF3 and AcrIIC1 can prevent target DNA cleavage. Using X-ray crystallography, AcrE1 was discovered to bind to the CRISPR associated Cas3.<ref>{{cite journal | vauthors = Pawluk A, Shah M, Mejdani M, Calmettes C, Moraes TF, Davidson AR, Maxwell KL | title = Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein | journal = mBio | volume = 8 | issue = 6 | date = December 2017 | pmid = 29233895 | pmc = 5727412 | doi = 10.1128/mBio.01751-17 | doi-access = free}}</ref> Likewise, biochemical and structural analysis of AcrIF3 showed its capacity of binding to Cas3 as a dimer so as to prevent the recruitment of Cas3 to the Cascade complex.<ref name="Bondy-Denomy_2015" /><ref>{{cite journal | vauthors = Wang J, Ma J, Cheng Z, Meng X, You L, Wang M, Zhang X, Wang Y | display-authors = 6 | title = A CRISPR evolutionary arms race: structural insights into viral anti-CRISPR/Cas responses | journal = Cell Research | volume = 26 | issue = 10 | pages = 1165–1168 | date = September 2016 | pmid = 27585537 | pmc = 5113301 | doi = 10.1038/cr.2016.103 }}</ref><ref>{{cite journal | vauthors = Wang X, Yao D, Xu JG, Li AR, Xu J, Fu P, Zhou Y, Zhu Y | display-authors = 6 | title = Structural basis of Cas3 inhibition by the bacteriophage protein AcrF3 | journal = Nature Structural & Molecular Biology | volume = 23 | issue = 9 | pages = 868–70 | date = September 2016 | pmid = 27455460 | doi = 10.1038/nsmb.3269 | s2cid = 6466590 }}</ref> Finally, thanks to biochemical and structural AcrIIC1 studies, it was found that it binds to the active site of the HNH endonuclease domain in Cas9, which prevents DNA from cleaving. Thus, it turns Cas9 into an inactive but DNA bound state.<ref name="Harrington_2017" />

== Applications == thumb|'''Phage therapy''' could be used against antibiotic resistance, as bacteriophages can kill bacteria, and cure an infection.

=== Reducing CRISPR-Cas9 off-target cuts === AcrIIA4 is one of the proteins responsible for the CRISPR-Cas9 system inhibition, the mechanism used in mammalian cells edition. Addition of AcrIIA4 in human cells avoids Cas9 interaction with the CRISPR system, reducing its ability to cut DNA. However, diverse studies have reached the conclusion that adding it in small proportions after the genome editing has been done, reduces the number of off-target cuts at the concrete sites in which Cas9 interacts, a thing that makes the whole system much more precise.<ref name = "Shin_2017" />

=== Avoiding ecological consequences === One of the main objectives of using CRISPR-Cas9 technology is eradicating diseases, some of which are found in disease vectors, such as mosquitoes. Anti-CRISPR proteins can impede gene drive, which could create uncertain and catastrophic consequences in ecosystems.<ref name="Zhang_2019">{{cite journal | vauthors = Zhang F, Song G, Tian Y | title = Anti-CRISPRs: The natural inhibitors for CRISPR-Cas systems | journal = Animal Models and Experimental Medicine | volume = 2 | issue = 2 | pages = 69–75 | date = June 2019 | pmid = 31392299 | pmc = 6600654 | doi = 10.1002/ame2.12069 }}</ref>

=== Detect presence of Cas9 in a sample === thumb|Phage therapy is a good alternative to the use of antibiotics, but some bacteria have CRISPR-Cas systems. Nevertheless, if phages had Acr proteins, they would inhibit the CRISPR-Cas immune system and infect the cell. At the end of the phage reproduction cycle, which takes place inside bacteria, new phages would be released, provoking the cell lysis. In order to know whether a certain bacterium synthesises Cas9, and therefore uses CRISPR-Cas9, or to detect accidental or not allowed use of this system, AcrIIC1 can be used. As the aforementioned protein binds to Cas9, a centrifugal microfluidic platform has been designed to detect it and determine its catalytic activity.<ref name="Zhang_2019" />

=== Phage therapy === Antibiotic resistance is a public health problem that is constantly increasing, because of the bad use of antibiotics. Phage therapy consists of the infection of bacteria using phages, which are much more specific and cause less side effects than antibiotics. Acrs could inhibit the CRISPR-Cas9 system of some bacteria and allow these phages to infect bacterial cells without being attacked by its immune system.<ref name="Zhang_2019" />

== See also == {{Div col|colwidth=16 em}}{{div col}} * CRISPR * CRISPR/Cas Tools * CRISPR gene editing * Gene knockout * Bacteriophage * Phage therapy * Genetics * Bacteria

{{div col end}}{{Div col end}}

== References == {{reflist}}

Category:Proteins Category:Genetic engineering Category:Genome editing