Abstract
Bacteria and bacteriophages have been involved in an evolutionary arms race for billions of years. The basis of the arms race results from bacteria evolving defense mechanisms to protect and prevent themselves from phage infection, while phages coevolve to evade these defense mechanisms. One of the mechanisms of defense bacteria use is the CRISPR-Cas systems, which destroy foreign genetic elements. Phages coevolved anti-CRISPR proteins, which are involved in inhibiting the interference stage in the CRISPR-Cas system. The discovery, mechanisms, and applications of anti-CRISPR proteins will be further discussed, with a focus on type I and type II systems.
Introduction
Bacteriophages (phages) are viruses that infect bacteria and are likely the most abundant biological entity on Earth1,2. Phages outnumber bacteria significantly, resulting in a considerably large amount of infections per second1,2. The effect of this interaction is an immense selective pressure on bacteria resulting in an evolutionary arms-race between phages and bacteria2,3. Bacteria have evolved various defense mechanisms to prevent and protect against infections, while viruses have coevolved to adapt to these defenses1,2,3. One of the mechanisms of defense bacteria have evolved is to mutate the receptor that the phage recognizes and binds to, the mutation results in the phage losing its ability to recognize the receptor; thus, infection is prevented2. Phages evolved the ability to escape this defense by recognizing the mutated receptors by modifying their receptor binding proteins or by binding to a new receptor2. The abortive infection mechanism can be used to prevent further infection of a bacterial population if the phage genome has entered the cytoplasm; the bacteria can trigger pre-mature cell death upon recognizing genes that trigger abortive infection systems1,2. Phages coevolved to alter genes to inhibit triggering the abortive infection system or to produce antitoxins2. The CRISPR-Cas system is an important defense mechanism that has evolved in bacteria and archaea, and is found abundantly across species of both domains1,2,4,5. The CRISPR-Cas system involves adaptive immunity to protect against phage infection1,2. The system is involved in cleaving foreign genomes such as the phage genome through restriction-modification systems1,4,5. There is a huge diversity in the CRISPR-Cas types; however, this review will focus on type I and type II CRISPR-Cas systems1,2. The CRISPR-Cas system is an effective mechanism to prevent infection; however, phages have counterattacked the system through anti-CRISPR proteins 2,3,4. The anti-CRISPR system has various mechanisms to inactivate the CRISPR-Cas interference complexes resulting in inhibition of the defense system while gaining the ability to infect 2,3,4,5.
CRISPR-Cas System
The CRISPR-Cas system in bacteria and archaea form an adaptive immune system against invasion by bacteriophages, plasmids, and other genetic elements1,2,4. The CRISPR-Cas defense mechanisms involve three key processes; adaption, expression, and interference1. The CRISPR-Cas system consists of the CRISPR array, which has palindromic direct repeats separated by spacers; the spacers are short repeats which are derived from the phage genome or other genetic elements1,2,3. The addition of spacers into the CRISPR gene locus is the adaption process1. The expression stage involves transcription of the CRISPR array results in CRISPR RNAs (crRNAs), which is assembled in association with Cas proteins1,2,3,6. The interference stages involve the CRISPR-Cas complex recognizing any complementary sequence to the spacer sequence, and upon recognition the virus is disabled2,6. The interference stage in both type I and type II systems target DNA sequences and require the spacer sequence on the crRNA to be completely complementary to the nucleotides of the protospacer adjacent motif (PAM) on the foreign genetic element for binding1,15,5. Initially, the only known mechanism of viruses evading the CRISPR-Cas system was to mutate the PAM sequence to protect against targeting2,4,15. However, a new system has been identified that counteracts the CRISPR-Cas system, and this system is called the anti-CRISPR system2,3,6.
Discovery of anti-CRISPR
The anti-CRISPR system was first identified in 2013 in phages that infect Pseudomonas aeruginosa6. The system was discovered through Pseudomonas aeruginosa phages, which were integrated into the bacteria as prophages and were expected to be destroyed by the CRISPR-Cas system as they possessed an active type I-F system6. However, some of the phages evaded destruction by the CRISPR-Cas system. This resulted in further analysis and genome sequence comparisons between phages that were destroyed to those that were not6. The genome analysis identified a genetic locus that encoded ten different genes, which all had unknown function. The ability to inhibit the CRISPR-Cas activity by the ten genes was tested and resulted in the identification of five genes (acrIF1 to acrIF5) that had inhibiting activity on the type I-F system3,6,8, 20,21. The anti-CRISPR protein families are highly diverse, such that the original protein sequences did not contain similar genomes, which made it difficult for the discovery of anti-CRISPR proteins3,6,9,20. However, the anti-CRISPR gene organization is highly conserved as each of the discovered phages had an anti-CRISPR associated 1 (aca1) protein, which was directly after the acrE/acrF genes7,9,20. This region led to the identification of more anti-CRISPR families. The anti-CRISPR genes are now classified as moron genes, which do not play a role in the life cycle of the virus, but they provide a significant evolutionary advantage4.
Type I System
The CRISPR-Cas type I system has multiple Cas proteins, which include Cas5, Cas7, and Cas8 proteins making up a complex called Cascade, but the Cas3 protein is the main effector protein7,10,5,20. The cascade complex binds to crRNA to identify the DNA target and recruits Cas3, which has exonuclease and helicase activity for the destruction of a target sequence. Currently, there are seven subtypes of type I CRISPR-Cas systems7,10,20. Pseudomonas aeruginosa can possess both type I-E and type I-F systems7,8. The type I-F anti-CRISPR proteins include AcrF1, AcrF2, and AcrF3. AcrF1 and AcrF2 are involved in direct binding to the Csy cascade complex, which results in inhibition of DNA binding8,5. The type I-E system is inhibited by anti-CRISPR proteins acrIE1 through acrIE4, which inactivate the interference step of the CRISPR-Cas system7,8,5. In 2014, Pawluk et al. stated that the anti-CRISPR proteins were specific to one system; thus, the type I-F anti-CRISPRs only inhibited the type I-F system and did not affect type I-E system9. However, in 2016, Pawluk et al. discovered AcrIF6 protein, which has dual specificity for both type I-E and type I-F systems11. These studies indicate that the knowledge of anti-CRISPR proteins is limited, and new studies are changing our views on the mechanisms of anti-CRISPR proteins.
Due to the diversity of anti-CRISPR proteins, there are a variety of mechanisms through it inhibits the CRISPR-Cas system. One of the most common mechanisms of action is disrupting the DNA binding through interaction with CRISPR interference proteins5. In the type-I-F CRISPR-Cas system, the anti-CRISPR protein AcrIF1 binds to the cascade proteins Cas7f, this binding result in inhibition as Cas7f is responsible for targeting DNA binding8,18,20. Inhibition of target cleavage is another mechanism used for CRISPR-Cas inhibition, and the mechanism involves inhibiting the nuclease activity 5,20. In the type I-F system, AcrIF3 binds to Cas3, which has nuclease activity, binding of the anti-CRISPR results in inhibition of nuclease activity and thus inhibition of the CRISPR-Cas system5,18. Another mechanism through anti-CRISPR proteins inhibit the CRISPR-Cas system is through the disruption of DNA binding by mimicking DNA. In type I anti-CRISPR AcrIF2 competes with DNA for DNA binding to the Cas proteins of type I-F system5.
Type II system and Cas9 Inhibition
The CRISPR-Cas type II system has the Cas9 protein, which is the single effector protein5. The Cas9 protein is involved in identifying DNA targets, destroying the target, spacer acquisition, and many other functions21. Currently, there are three subtypes of the type 2 CRISPR-Cas systems identified5,20. CRISPR-Cas9 is simple as it requires one protein for its function. CRISPR-Cas 9 has many applications, which include but are not limited to genome editing and localization of genes.
The type II inhibitory mechanisms of CRISPR-Cas system are similar to the type I mechanisms. The first anti-CRISPR proteins identified were AcrIIC1, AcrIIC2, AcrIIC3 in the type II-C system in Neisseria meningitides5,20,21. The proteins are all involved in inhibition of the type II CRISPR-Cas9 system. The AcrIIC1 protein is similar to AcrF3, as they are both involved in blocking cleavage by binding to the Cas9 protein, which has nuclease activity5,11,21. The AcrIIC3 protein is involved in inducing dimerization of Cas9, which inhibits DNA target recognition5,20. In Listeria monocytogenes four new families were discovered; AcrIIA1, AcrIIA2, AcrIIA3, and AcrA45,20. The AcrIIA4 has similar functions to AcrF2, as it is involved in mimicking double-stranded DNA and can block the PAM binding site5,20.
Applications of anti-CRISPR proteins
Anti-CRISPR proteins can serve as an essential component of genome editing mediated by the CRISPR-Cas9 system. As previously stated, the recently discovered anti-CRISPR proteins have the ability to inhibit cas9 activity11. This natural ability to inhibit cas9 function could be essential to the gene-editing process. Although CRISPR-Cas9 is effective, it does have its downsides, which include many errors and off-target DNA cleavage. The anti-CRISPR protein can serve as an inhibitor of Cas9, which could limit the number of errors made by Cas914,16. A recent study discovered that the addition of the anti-CRISPR protein AcrIIA4 to human cells before genome editing results in complete inhibition of CRISPR-Cas916. Whereas the addition of AcrIIA4 following genome editing results in a reduction of off-target DNA cutting16. Anti-CRISPR can also be potentially used as anti-microbial agents2.
Conclusion
Anti-CRISPR proteins are the counterattack to the adaptive immunity bacteria have gained through CRISPR-Cas systems. The anti-CRISPR proteins are diverse and have many mechanisms of function, which include disruption of DNA binding, inhibition of target cleavage, and DNA mimicry. The type I system has several proteins that make up the cascade complex, and its crucial protein is Cas3. Whereas the type II system has Cas9, which can carry out many functions. The diversity and complexity of anti-CRISPR proteins are vast, and the knowledge is still limited as it is a recent discovery. Future studied of anti-CRISPR may reveal functions and mechanisms that could enhance the genome editing process making it more effective and accurate. Future studies of anti-CRISPR should be conducted to answer the possible question if bacteria have evolved the ability to evade anti-CRISPR proteins.
2019-10-29-1572309599