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Project titleStudy of adaptation and interference by natural Type III CRISPR-Cas systems of bacterium T. thermophilus

AffiliationAutonomous Non-Profit Organization for Higher Education "Skolkovo Institute of Science and Technology",

KeywordsCRISPR-Cas systems, Type III, CSM, CMR, adaptation, interference, Cas1, bacteriophages, bacterial immunity, resistance, thermophilic bacteria

Annotation
CRISPR-Cas systems are adaptive and heritable immunity systems of prokaryotes that protect bacteria and archaea cells from viruses and prevent the penetration of plasmids. CRISPR-Cas systems consist of CRISPR arrays and associated cas genes. The CRISPR array is a special place in the genome where identical repeats are interspersed with unique segments called "spacers". In general, the mechanism of action of all CRISPR-Cas systems can be divided into 3 stages. The first is called adaptation. Adaptation is the acquisition of immune memory by embedding in the CRISPR array spacers (fragments of foreign DNA). The second stage is the expression and maturation of all components of the systems necessary for protection of the cell. The third one is the interference. Interference is a specific recognition and a destruction of an invader. To date, 6 different Types of systems are defined: I to VI. The classification is based on the protein composition of ribonucleoprotein complexes, the so-called effector complexes, providing the stage of interference. The uniqueness of the Type III CRISPR-Cas system is that it is the only Type of all currently known, which is characterized by a transcriptional-dependent mechanism of action, i.e. immunity can be provided only in the case of target transcription. Effector complex has both and RNAse and DNAse activity only in the Type III systems. As a consequence, interference mechanism of Type III CRISPR-Cas systems is the most complex one. It includes a large number of sub-stages, precisely coordinated with each other. All sub-stages require the presence of many protein components, both as part of the effector complex and outside it. As of today, there are still many open questions regarding both the stages of adaptation and interference. This project involves the solution of two main challenges. First, the project will investigate the adaptation process in the laboratory strain of thermophilic bacterium Thermus thermophilis Hb27, which has active Type III systems, as in the conditions of infection with two different bacteriophages capable of infecting this strain, and in the conditions of transformation of cells with plasmid. This task is extremely urgent, because today only one laboratory in the world has managed to create conditions for the observation of adaptation in bacterium (Marinomonas mediterranea) with Type III CRISPR-Cas systems. It should be noted that M. mediterranea encodes the key adaptation protein Cas1 fused with the reverse transcriptase domain, allowing to acquire spacers from RNA. Thermus thermophilis Hb27 does not have reverse transcriptase in the strain. However, preliminary data obtained in our laboratory have shown that adaptation by Type III system in T. thermophilus Hb27 is possible. This indicates a fundamentally different mechanism for the acquisition of spacers. For this project we will conduct a high-throughput sequencing to analyze a large amount of acquired spacers in a case of interaction of CRISPR-Cas systems with each of the phages and a plasmid. In addition, mutant strains with gene deletion, presumably necessary for the adaptation process, will be obtained by via homologous recombination. After this experiments on absence of adaptation will be carried out on these strains. The second task is to determine the efficiency and characteristics of the interference. We will validate the ability of cells that have acquired a spacer to resist infection by the corresponding phages or plasmid transformation. We will compare the effectiveness of defense in the case of acquisition of spacers from different regions for each of the phages/plasmid. In addition, the degradation of the DNA target will be studied by quantitative PCR method and high-throughput sequencing. The relevance of this problem is also high. At the moment the world scientific community has found that the effector complex of Type III systems does have nuclease activity against single-stranded DNA, however, the details of the of DNA target cleavage are unknown.

Expected results
As a result of this project, it is expected for the first time in the world to characterize the laboratory-detected adaptation in the bacterial CRISPR-Cas type III system in cells encoding the main adaptation protein Cas1 without the reverse transcriptase domain. In addition, high-throughput sequencing analysis of DNA-target during the stage of interference in the resistant strains obtained in the course of adaptation will be performed for the first time for Type III systems. These results will bring a greater understanding of the functioning of Type III CRISPR-Cas systems, which is not only interesting and important in itself, but also will bring practical benefits in light of the possibility of using endogenous CRISPR-Cas systems for editing genomes and post-transcriptional regulation of gene expression. Despite the complexity and multi-component structure, Type III CRISPR-Cas systems may be used for genomic editing and regulation of gene expression directly in organisms, in the genome of which they are encoded.

Annotation of the results obtained in 2020
The main part of research work during the second year of the project was devoted to studying the action of CRISPR-Cas Type III systems under conditions of infection with phiKo bacteriophage. phiKo is the second of two available bacteriophages infecting our model organism - the thermophilic bacterium T. thermophilus HB27c. phiKo belongs to Tectiviridae family, while the phiFa phage, which was studied in the first year of the study, is a member of the Siphoviridae family. Comparative analysis of the results obtained during infection of cells with phiFa and phiKo allows us to conclude that some of the revealed features of Type III CRISPR-Cas systems are features of Type III CRISPR-Cas defense system, and some are a consequence of interaction with a specific foreign agent. Thus, we have shown that 1) the uneven distribution of spacers along the DNA chains is a consequence of the selection of cells with protective spacers, and there are no special mechanisms for selection / insertion of spacers into CRISPR arrays in the "right" direction 3) CRISPR-Cas systems Type III do not work on the principle of suicidal death. It was shown that, in contrast to phiFa, spacers targeting transcripts which are synthesized at any stage of infection, can protect bacterial cells. In addition to these results, temporal classes of phiKo genes were identified, and it was shown that Cas4 proteins do not affect the adaptation of Type III CRISPR-Cas systems.

Publications

1. Daria Artamonova, Karyna Karneyeva, Sofia Medvedeva, Evgeny Klimuk, Matvey Kolesnik, Anna Yasinskaya, Aleksei Samolygo, Konstantin Severinov Spacer acquisition by Type III CRISPR–Cas system during bacteriophage infection of Thermus thermophilus Nucleic Acids Research, - (year - 2020) https://doi.org/10.1093/nar/gkaa685

2. Kolesnik M., Karneeva K., Artamonova D., Severinov K. Type III CRISPR-Cas systems: the dissection of the most complex prokaryotic immune system Biochemistry (Moscow), - (year - 2021)

3. - В Сколтехе узнали, как бактерии собирают «разведданные» о бактериофаге Naked Science, - (year - )

Annotation of the results obtained in 2019
During the first year, the properties of adaptation and interference were studied for Type III CRISPR-Cas systems of bacterium T. thermophilus in the course of infection with siphovirus phiFa. The optimal conditions for adaptation were selected. Using high-throughput sequencing technology, a pool of spacers acquired by Type III CRISPR arrays during phage infection was characterized. It was shown that the observed bias for the distribution of acquired spacers over the phage genome is a consequence of counter-selection and accumulation of cells with spacers that can provide protection, and not a feature of the Type III adaptation machinery. We isolated seven strains, acquired 1-2 new spacers in their Type III CRISPR arrays, and each of these strains was resistant to phiFa. A method has been developed for introducing any desirable spacers into cells by transformation of cells with a plasmid carrying an artificial mini-array. We have demonstrated that only spacers originated from the end of the phiFa genome which first enters the cell are able to make cells resistant to phiFa. Spacers targeting phiFa genes belonging to the "middle" and "late" temporal classes are not able to provide protection. We have shown that phiFa can escape the action of the CRISPR-Cas Type III system by accumulation of deletions in the protospacer disturbing the complementarity between the spacer and protospacer. Deletions in the genomes of escaper phages can be both long ( up to several hundred nucleotides) or short. When essential genes (such as the gene coding RNA polymerase) are targeted, phage can escape Type III CRISPR-Cas system action by short deletions with a length multiple of three nucleotide to preserve a bona fide reading frame. To investigate the state of phage DNA during infection of phiFa resistant strain, total DNA was isolated from the infected culture and was analyzed using high-throughput sequencing. No extensive gaps (like these which is typical for Type I CRISPR-Cas systems interference process) along phiFa DNA were detected, but it was found that amount of phage DNA did not increase with the course of infection of the resistant strain while in the case of control WT strain it did increased significantly.

Publications