EMD-35926

Single-particle
2.91 Å
EMD-35926 Deposition: 13/04/2023
Map released: 27/09/2023
Last modified: 09/10/2024
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links

EMD-35926

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

EMD-35926

Single-particle
2.91 Å
EMD-35926 Deposition: 13/04/2023
Map released: 27/09/2023
Last modified: 09/10/2024
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Sample Organism: Sulfoacidibacillus thermotolerans
Sample: AsCas12f-YHAM-sgRNAS3-5v7-target DNA
Fitted models: 8j1j (Avg. Q-score: 0.511)

Deposition Authors: Hino T, Omura NS, Nakagawa R, Togashi T, Takeda NS, 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
An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.
PUBMED: 37776859
DOI: doi:10.1016/j.cell.2023.08.031
ISSN: 1097-4172
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.