(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)
