An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis

Tomohiro Hino¹, Satoshi N Omura², Ryoya Nakagawa², Tomoki Togashi³, Satoru N Takeda², Takafumi Hiramoto⁴, Satoshi Tasaka¹, Hisato Hirano², Takeshi Tokuyama⁵, Hideki Uosaki⁵, Soh Ishiguro⁶, Madina Kagieva⁶, Hiroyuki Yamano⁷, Yuki Ozaki⁷, Daisuke Motooka⁸, Hideto Mori⁹, Yuhei Kirita¹⁰, Yoshiaki Kise¹¹, Yuzuru Itoh², Satoaki Matoba¹, Hiroyuki Aburatani¹², Nozomu Yachie¹³, Tautvydas Karvelis¹⁴, Virginijus Siksnys¹⁴, Tsukasa Ohmori¹⁵, Atsushi Hoshino¹⁶, Osamu Nureki¹⁷

Affiliations

¹ Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.
² Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.
³ Department of Biochemistry, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan; Department of Clinical Laboratory Science, Division of Health Sciences, Graduate School of Medical Science, Kanazawa University, Ishikawa 920-0942, Japan.
⁴ Department of Biochemistry, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan.
⁵ Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan.
⁶ School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, BC V6S 0L4, Canada.
⁷ Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
⁸ Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Suita, Osaka 565-0871, Japan.
⁹ Institute for Advanced Biosciences, Keio University, Yamagata 997-0035, Japan; Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa 252-0882, Japan; Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Osaka, 565-0871, Japan.
¹⁰ Department of Nephrology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.
¹¹ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan; Curreio, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
¹² Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan.
¹³ School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, BC V6S 0L4, Canada; Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Osaka, 565-0871, Japan; Synthetic Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan.
¹⁴ Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius, Lithuania.
¹⁵ Department of Biochemistry, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan; Center for Gene Therapy Research, Jichi Medical University, Tochigi 329-0498, Japan. Electronic address: tohmori@jichi.ac.jp.
¹⁶ Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan. Electronic address: a-hoshi@koto.kpu-m.ac.jp.
¹⁷ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan. Electronic address: nureki@bs.s.u-tokyo.ac.jp.

PMID: 37776859
DOI: 10.1016/j.cell.2023.08.031

Free article

An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis

Tomohiro Hino et al. Cell. 2023.

Free article

. 2023 Oct 26;186(22):4920-4935.e23.

doi: 10.1016/j.cell.2023.08.031. Epub 2023 Sep 29.

Authors

Affiliations

¹ Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.
² Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.
³ Department of Biochemistry, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan; Department of Clinical Laboratory Science, Division of Health Sciences, Graduate School of Medical Science, Kanazawa University, Ishikawa 920-0942, Japan.
⁴ Department of Biochemistry, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan.
⁵ Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan.
⁶ School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, BC V6S 0L4, Canada.
⁷ Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
⁸ Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Suita, Osaka 565-0871, Japan.
⁹ Institute for Advanced Biosciences, Keio University, Yamagata 997-0035, Japan; Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa 252-0882, Japan; Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Osaka, 565-0871, Japan.
¹⁰ Department of Nephrology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.
¹¹ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan; Curreio, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
¹² Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan.
¹³ School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, BC V6S 0L4, Canada; Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Osaka, 565-0871, Japan; Synthetic Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan.
¹⁴ Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius, Lithuania.
¹⁵ Department of Biochemistry, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan; Center for Gene Therapy Research, Jichi Medical University, Tochigi 329-0498, Japan. Electronic address: tohmori@jichi.ac.jp.
¹⁶ Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan. Electronic address: a-hoshi@koto.kpu-m.ac.jp.
¹⁷ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan. Electronic address: nureki@bs.s.u-tokyo.ac.jp.

PMID: 37776859
DOI: 10.1016/j.cell.2023.08.031

Abstract

SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.

Keywords: CRISPR-Cas; animal experiments; cryo-EM; deep mutational scanning; gene therapy; genome editing; iPS cells.

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Conflict of interest statement

Declaration of interests T.H., S.N.O., R.N., Y.K., S.M., T.O., A.H., and O.N. have filed a patent application related to this work.

Comment in

Engineering a compact genome-editing tool.
Crunkhorn S. Crunkhorn S. Nat Rev Drug Discov. 2023 Dec;22(12):953. doi: 10.1038/d41573-023-00170-1. Nat Rev Drug Discov. 2023. PMID: 37884601 No abstract available.

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An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis

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An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis

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Abstract

Conflict of interest statement

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