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Uncovering viral sabotage of host CRISPR-Cas immune systems

Periodic Reporting for period 1 - Anti-CRISPR (Uncovering viral sabotage of host CRISPR-Cas immune systems)

Reporting period: 2018-01-15 to 2020-01-14

(1) Research goals
1. Identification of anti-CRISPR (Anti-CRISPR, ACR) genes of bacteriophage through the high-throughput method
2. Biophysical characterization of the molecular mechanism of the identified ACR protein using single-molecule fluorescence microscopy

(2) Research background
Prokaryotic viruses, which are representatives of bacteriophage, are one of the most threatening elements for prokaryotic survival, and the intense arms race between bacteriophage and host prokaryotic organisms has evolved to have more powerful attacks and defenses against each other. If a bacteriophage can express an anti-CRISPR protein, it can neutralize the host's CRISPR-Cas immune system and provide an evolutionary advantage to maximize the proliferation of the bacteriophage. These ACRs are highly likely to have evolved very specifically depending on the host-virus relationship, so ACRs expressed by different bacteriophages will each have a wide variety of structural and functional characteristics, and thus the host's CRISPR-Cas immune system In response to this, it would have evolved in various ways. Since the diversity of the CRISPR-Cas immune system can be thought to have occurred competitively by the bacteriophage immune evasion strategy, researches on ACR itself will be a good way to further deepen functional and structural research on not only biological evolution but also the CRISPR-Cas immune system.

(3) Research scopes and aims
The ACR of Bacteriophage was planned to be newly discovered and molecular mechanisms related to the identified ACR were planned to be studied. In particular, this trainee has focused on new ACR protein discovery studies on the Cas9 protein-mediated type II CRISPR-Cas system, which is known to be the most important defense against lactic acid bacteria. LAB is a bacterium that is very important industrially (particularly fermentation industry), and infection control for bacteriophage is one of the important areas for industrial stable and efficient production management. This researcher has aimed to establish a high-throughput research method consisting of military genomics, bioinformatics, and protein mass spectrometry, utilizing past research experiences for this study. After the identification of the new ACR, the molecular mechanism of the ACR was planned to be elaborated using the most advanced single-molecule fluorescence analysis method, the largest research tool of the institute's training institution. This single-molecule analytical approach is considered as a good way to overcome the limitations of the traditional biochemical approach. As one of the main causes of bacteriophage infection, which cannot be completely controlled by the present technology, the presence of a bacteriophage encoding the ACR gene is considered as a major candidate. If the final result of the study is applied to the fields of science, technology, and society, it is thought that it will make a significant contribution to establishing a new genetic modification strategy. The detailed findings are expected to provide important application potential for bacteriophage therapy, food industry, agriculture, and genetic modification techniques, while also providing new insights into the host-viral arms race.

(4) Contingency plans
- Although there is an optimistic prospect for the ACR protein identification study for the proposed Type II CRISPR-Cas, if it fails for a given period, backup projects have been planned to be studied.
- Studies of the mechanism of ACRs that have been already identified by other research groups.
- Studies of the mechanisms of CRISPR-Cas adaptation (to establish the background knowledge to understand which points in CRISPR-Cas adaptation might be targeted by ACRs)
During the period of collecting various methods and research articles to carry out this study, the research contents with the same purpose as this study have been published in several papers by different research groups, like below.
- Cell. 2016 Dec 15;167(7):1829-1838.
- Cell. 2017 Jan 12;168(1-2):150-158.
- Nature. 2017 Jun 15;546(7658):436-439.
- Mol Cell. 2017 Jul 6;67(1):117-127
- Sci Adv. 2017 Jul 12;3(7):e1701620
- Nat Microbiol. 2017 Aug 7.
- Cell. 2017 Sep 7;170(6):1224-1233.

Therefore, it has been decided that the study that identified ACR of a new type of Cas9 protein, which was the primary goal of this study, has no meaning.As the first proposed contingency plan, a joint research proposal by Blake Wiedenheft (Montana State University) received a joint research proposal on the molecular mechanism through the single-molecule biophysical method of Pseudomonas aeruginosa ACR. Collaborating with a Bachelor student Rochelle Niemeijer, an ACR, acrIE, has been studied.

For the second contingency plan, the molecular mechanism of spacer acquisition (CRISPR-Cas adaptation) has been successfully studied and published in Nature on 19 Feb in 2020 (Selective loading and processing of prespacers for precise CRISPR adaptation, Nature volume 579, pages141–145(2020),
(1) The study of the molecular mechanism of AcrIE: Research methods and contents related to ACR published to date are mainly related to the identification of new types of ACR or to reveal the structure of the identified ACR alone or with the target Cas9 protein. Therefore, until now, studies on molecular mechanisms in dynamic situations using single-molecule fluorescent FRET have not been published. Not only is the research content expected to be published, but it is also expected to provide fundamental information that will revolutionize the development of DNA-related technologies related to Cas9 and other types of CRISPR-Cas systems.
(2) Impact of the study of the molecular mechanism of CRISPR-Cas adaptation: Several groups have harnessed the nucleic-acid-acquisition abilities of Cas1–Cas2 to develop new techniques for recording nucleic acids in cellular contexts. Cas1–Cas2-based recording techniques allow cellular events in prokaryotes to be captured in chronological order. Our results may help in developing the next generation of Cas1–Cas2 recorders that are more efficient at capturing information. Moreover, our findings may also enable a Cas1–Cas2-based recording system to be developed in eukaryotes, which has not previously been reported. This system can be converted into a 'DNA recorder': a kind of biological log that keeps track of what happens in a cell, and in which you can, for example, read how a tumor has developed over time. The first versions of such a DNA recorder have already been built, but are inefficient and do not work in human cells. The new fundamental knowledge about bacterial memory formation can be used to develop the next generation of DNA recorders.
Nature volume 579, pages141–145(2020)