InaRIS Fellow (2021-2030)

Hiroshi Nishimasu

Professor,Research Center for Advanced Science and Technology, The University of Tokyo*Profile is at the time of the award.

2021InaRISBiology & Life sciences

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Research topics: Exploration of new RNA-dependent enzymes

Keyword

Summary: The RNA-guided DNA-cleaving enzyme Cas9 from the microbial CRISPR-Cas adaptive immune system has been extensively studied and applied to innovative technologies such as genome editing. In addition to Cas9, extremely diverse Cas enzymes exist in nature, but their functions remain largely unknown. In this study, we will explore and elucidate the functions and structures of new Cas enzymes. Furthermore, we will try to develop innovative technologies that revolutionize the life sciences.

Message from fellow

Last year I opened my own laboratory, and this generous grant will allow me to devote my time to research. I am very grateful for support from the Inamori Research Institute for Science (InaRIS) for the next 10 years. The timing is extremely fortunate as I am at a turning point in my research life. I would like to engage in curiosity-driven research about the action mechanisms of proteins and nucleic acids as my research lifework.

InaRIS Fellow Profile Video

Reports

Three-Year AcheivementsCitation

We determined the cryo-EM structures of several novel Cas enzymes, including Cas12c2, Cas12f, and Cas13bt3, elucidating their diverse mechanisms of action. In 2020, I established my own lab at The University of Tokyo and elucidated the cryo-EM structures of the new Cas enzyme Cas7-11-Csx29 and IscB, the ancestor of Cas9. In addition, I initiated research on DNA recombinases, determining the cryo-EM structures of the serine recombinase Bxb1 and a unique RNA-guided IS110 recombinase. These studies provide mechanistic insights into DNA recombination and establish a foundation for advancing genome engineering technologies.

In the CRISPR-Cas adaptive immune system, diverse Cas enzymes are responsible for defense against foreign nucleic acids and form ribonucleoprotein complexes with their cognate guide RNA to recognize and cleave target nucleic acids (DNA or RNA). Using cryo-electron microscopy, we have determined the structures of several novel Cas enzymes, including Cas12c2 (Kurihara et al., Mol Cell 2022), Cas12f (Takeda et al., Mol Cell 2021), and Cas13bt3 (Nakagawa et al., Mol Cell 2022), elucidating their distinct mechanisms of action. In 2020, I established a laboratory at The University of Tokyo and have been promoting structural and functional studies of the novel Cas enzyme complex Cas7-11-Csx29 and IscB, the likely ancestor of Cas9 (Kato et al., Cell 2022; Kato et al., Science 2022; Kato et al., Nat Commun 2022). We also initiated research on DNA recombinase such as the serine recombinase Bxb1 and the novel bridge RNA-guided IS110 family recombinase.

In the type III-E CRISPR-Cas system, Cas7-11 functions as an RNA-guided RNA nuclease that specifically cleaves single-stranded RNA targets at two sites. While Cas7-11 could be used as a highly specific RNA-targeting tool with low cytotoxicity, its RNA cleavage mechanism was enigmatic. Furthermore, the III-E type CRISPR-Cas locus encodes uncharacterized proteins, such as Csx29, Csx30, Csx31, and RpoE, suggesting that these proteins collaborate in antiviral defense. We determined the cryo-EM structure of the Cas7-11-guide RNA-target RNA complex and elucidated the Cas7-11-mediated RNA cleavage mechanism (Kato et al., Cell 2022). The structure revealed that Cas7-11 comprises four Cas7 domains (Cas7.1-Cas7.4), a Cas11 domain, an INS domain, a CTE domain, and four linker regions. Our biochemical experiments showed that Cas7-11 cleaves the phosphodiester bonds between bases 3-4 and 9-10 of the target RNA. Based on the structural information, we engineered a compact Cas7-11 variant (Cas7-11S), which can be efficiently delivered to target tissues as a knockdown tool (Kato et al., Cell 2022).

Furthermore, our biochemical analyses revealed that when the target RNA binds to the Cas7-11-guide RNA-Csx29 complex, Csx30 is cleaved by Csx29 into two fragments (Kato et al., Science 2022). The cryo-EM structures of the Cas7-11-guide RNA-Csx29 complex and the Cas7-11-guide RNA-Csx29-target RNA complex revealed that the binding of target RNA to the complex induces a structural change in the Csx29 active site, allowing Csx30 recognition and cleavage. These findings demonstrated that the Cas7-11-Csx29 complex is an unprecedented RNA-guided nuclease-protease complex with both RNA and Csx30 cleavage activities. Furthermore, we found that overexpression of Csx30 in Escherichia coli inhibits bacterial growth and that Csx30, Csx31, and RpoE form a complex, suggesting that in the type III-E system, the Cas7-11-Csx29 complex cleaves viral RNA and endogenous Csx30, thereby inhibiting the growth of infected cells through RpoE-mediated transcriptional regulation (Kato et al., Science 2022).

The RNA-guided DNA nuclease Cas9 is widely used in genome editing. Recent studies have indicated that Cas9 evolved from the transposon-encoded RNA-guided DNA nuclease IscB. However, the details of this molecular evolution remain unclear. To explore this, we determined the cryo-EM structure of the IscB-guide RNA-target DNA complex. A structural comparison of IscB and Cas9 suggested that the acquisition of protein domains and the miniaturization of the guide RNA contributed to the molecular evolution of CRISPR-Cas9 (Kato et al., Nat Commun 2022).

Recombinases are the most abundant proteins in nature and are classified into transposases and integrases, performing diverse functions across species from viruses to higher eukaryotes. The phage-derived large serine recombinase Bxb1 has been utilized in genome engineering technologies, as it catalyzes recombination between two DNA molecules with specific sequences (attP and attB). Serine recombinases have been studied since the 1980s, and a DNA recombination mechanism involving rotation has been proposed. However, their detailed mechanisms remain unclear. To address this, we determined the cryo-EM structure of the Bxb1-DNA complex, providing visual evidence for its unique DNA recombination mechanism.

We also investigated the structure and function of a novel RNA-guided IS621 recombinase, which belongs to the IS110 family of recombinases. Our biochemical and cellular analyses revealed that the IS621 recombinase forms a complex with a bridge RNA and catalyzes recombination between donor DNA and target DNA that have sequences complementary to the guide segments in the bridge RNA. Furthermore, we determined the cryo-EM structure of the IS621 recombinase-bridge RNA-donor DNA-target DNA complex, elucidating the molecular mechanism of unprecedented RNA-guided DNA recombination. IS110 family recombinases are expected to be used as new genome engineering tools, and this structural information will serve as a foundation for the molecular engineering of IS110 recombinases.

CRISPR-Cas is a “robust” system responsible for acquired immune system in bacteria. When a bacterium is exposed to a viral infection, part of the viral sequence is incorporated into the CRISPR region of the bacterial genome, and when the same virus re-invades, the Cas enzyme uses the “memory” of viral sequence stored on CRISPR to cleave the invader’s genome. One of the Cas enzymes, Cas9, is an RNA-dependent DNA cleaving enzyme that uses an arbitrary RNA sequence as a template (guide RNA) to cleave the target DNA sequence at any desired point. Genome editing using CRISPR-Cas9 is a revolutionary technology that enables precise modification of genome sequences, the “blueprint of life”, and offers a wide range of applications from basic molecular biology research to drug discovery, gene therapy, and crop breeding. It is also fresh in our minds that it was the subject of last year’s Nobel Prize in Chemistry.

Dr. Nishimasu has made remarkable breakthroughs in understanding and applying the structure and function of the CRISPR-Cas system from the viewpoint of structural biology. For example, he was the first in the world to determine the crystal structure of the Streptococcus pyogenes Cas9-guide RNA-target DNA complex, which is widely used for genome editing. Furthermore, through the structure-guided protein engineering, he successfully redesigned the three-dimensional structure of Cas9 based on the structure he discovered and extended the range of application of genome editing technology fourfold. In addition, through collaboration with experts in single-molecule observation, he has succeeded in capturing a movie of the process of DNA cleavage by Cas9.

However, Cas enzymes are diverse, and there are many other types of Cas proteins besides Cas9. The function of many Cas enzymes is still not well understood. Recently, Dr. Nishimasu used cryo-electron microscopy to determine the structure of the guide RNA-target DNA complex of Cas12f, the smallest known Cas enzyme. Through structure-function analyses of Cas12f, he revealed a completely new mode of action of Cas enzyme.

Dr. Nishimasu’s research proposal aims to elucidate the operating principles of these Cas enzymes, whose functions are unknown, by utilizing his strengths in structural analysis of protein-RNA complexes and enzymological approaches. Furthermore, by teaming up with experts in marine metagenomics, he aims to search for completely new types of Cas enzymes from microorganisms that grow “flexibly” and “robustly” in extreme conditions such as the deep sea, and to elucidate their structures and functions. Microorganisms that adapt to and grow in extreme environments such as high temperature and high pressure are the frontier for the search for new enzymes: A great example includes Taq polymerase from a thermophilic bacterium has brought about the generalization of polymerase chain reaction (PCR) and innovation in research and diagnosis in medical biology.

Dr. Nishimasu is a rising star in structural biology who has just launched his own laboratory in the summer of 2020. Therefore, with the 10-year support of the InaRIS Fellowship, Dr. Nishimasu can pursue his bold and innovative ideas to advance the world of Cas enzymes . Furthermore, we hope that the fellowship will lead to the discovery of novel protein-RNA complex enzymes beyond Cas enzymes, and contribute to new developments in biology through the elucidation of their structures and operation mechanisms.