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

Animals Humans Mice CRISPR-Cas Systems Cryoelectron Microscopy Gene Editing Mutation Genetic Therapy
CRISPR-Cas animal experiments cryo-EM deep mutational scanning gene therapy genome editing iPS cells

Journal

Cell
ISSN: 1097-4172
Titre abrégé: Cell
Pays: United States
ID NLM: 0413066

Informations de publication

Date de publication:
26 10 2023
Historique:
received: 21 04 2023
revised: 27 06 2023
accepted: 23 08 2023
medline: 30 10 2023
pubmed: 1 10 2023
entrez: 30 9 2023
Statut: ppublish

Résumé

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.

Identifiants

DOI: 10.1016/j.cell.2023.08.031 PMID: 37776859
pubmed: 37776859
pii: S0092-8674(23)00963-7
doi: 10.1016/j.cell.2023.08.031
pii:
doi:

Types de publication

Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

4920-4935.e23

Informations de copyright

Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

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.

Auteurs

Tomohiro Hino (T)

Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.

Satoshi N Omura (SN)

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

Ryoya Nakagawa (R)

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

Tomoki Togashi (T)

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.

Satoru N Takeda (SN)

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

Takafumi Hiramoto (T)

Department of Biochemistry, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan.

Satoshi Tasaka (S)

Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.

Hisato Hirano (H)

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

Takeshi Tokuyama (T)

Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan.

Hideki Uosaki (H)

Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi 329-0498, Japan.

Soh Ishiguro (S)

School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, BC V6S 0L4, Canada.

Madina Kagieva (M)

School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, BC V6S 0L4, Canada.

Hiroyuki Yamano (H)

Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.

Yuki Ozaki (Y)

Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.

Daisuke Motooka (D)

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.

Hideto Mori (H)

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.

Yuhei Kirita (Y)

Department of Nephrology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.

Yoshiaki Kise (Y)

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.

Yuzuru Itoh (Y)

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

Satoaki Matoba (S)

Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.

Hiroyuki Aburatani (H)

Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan.

Nozomu Yachie (N)

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.

Tautvydas Karvelis (T)

Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius, Lithuania.

Virginijus Siksnys (V)

Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius, Lithuania.

Tsukasa Ohmori (T)

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.

Atsushi Hoshino (A)

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.

Osamu Nureki (O)

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.

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Classifications MeSH

questionsmedicales.fr › Eucaryotes › Animaux › An AsCas12f-based compact genome-editing tool...
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Primates › Haplorhini › Catarrhini › Hominidae › Humains › An AsCas12f-based compact genome-editing tool...
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Rodentia › Muridae › Murinae › Souris › An AsCas12f-based compact genome-editing tool...
questionsmedicales.fr › Phénomènes génétiques › Régulation de l'expression des gènes › Épigenèse génétique › Extinction de l'expression des gènes › Systèmes CRISPR-Cas › An AsCas12f-based compact genome-editing tool...
questionsmedicales.fr › Techniques d'investigation › Microscopie électronique › Cryomicroscopie électronique › An AsCas12f-based compact genome-editing tool...
questionsmedicales.fr › Techniques d'investigation › Techniques génétiques › Génie génétique › Édition de gène › An AsCas12f-based compact genome-editing tool...
questionsmedicales.fr › Phénomènes génétiques › Variation génétique › Mutation › An AsCas12f-based compact genome-editing tool...
questionsmedicales.fr › Techniques d'investigation › Techniques génétiques › Génie génétique › Thérapie génétique › An AsCas12f-based compact genome-editing tool...