8J1J

Cryo-EM structure of the AsCas12f-YHAM-sgRNAS3-5v7-target DNA

Summary for 8J1J

• Entry DOI: 10.2210/pdb8j1j/pdb
• EMDB information: 35926
• Descriptor: Transposase IS605 OrfB C-terminal domain-containing protein, DNA (38-MER), RNA (118-MER), ... (6 entities in total)
• Functional Keywords: crispr-cas, rna binding protein-dna complex, rna binding protein, rna binding protein-dna-rna complex, rna binding protein/dna/rna
• Biological source: Sulfoacidibacillus thermotolerans; More
• Total number of polymer chains: 5
• Total formula weight: 161784.55

Authors

Hino, T.,Omura, N.S.,Nakagawa, R.,Togashi, T.,Takeda, N.S.,Hiramoto, T.,Tasaka, S.,Hirano, H.,Tokuyama, T.,Uosaki, H.,Ishiguro, H.,Yamano, H.,Ozaki, Y.,Motooka, D.,Mori, H.,Kirita, Y.,Kise, Y.,Itoh, Y.,Matoba, S.,Aburatani, H.,Yachie, N.,Siksnys, V.,Ohmori, T.,Hoshino, A.,Nureki, O. (deposition date: 2023-04-13, release date: 2023-09-27, Last modification date: 2024-10-09)

Primary citation

Hino, T.,Omura, S.N.,Nakagawa, R.,Togashi, T.,Takeda, S.N.,Hiramoto, T.,Tasaka, S.,Hirano, H.,Tokuyama, T.,Uosaki, H.,Ishiguro, S.,Kagieva, M.,Yamano, H.,Ozaki, Y.,Motooka, D.,Mori, H.,Kirita, Y.,Kise, Y.,Itoh, Y.,Matoba, S.,Aburatani, H.,Yachie, N.,Karvelis, T.,Siksnys, V.,Ohmori, T.,Hoshino, A.,Nureki, O.
An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.
Cell, 186:4920-4935.e23, 2023

PubMed 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.

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

Experimental method

ELECTRON MICROSCOPY (2.91 Å)