EMD-35926
Cryo-EM structure of the AsCas12f-YHAM-sgRNAS3-5v7-target DNA
EMD-35926
Single-particle2.91 Å
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Map released: 27/09/2023
Last modified: 09/10/2024
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
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
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An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.
Hino T,
Omura SN,
Nakagawa R,
Togashi T,
Takeda SN,
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
(2023) Cell , 186 , 4920 - 4935.e23
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(2023) Cell , 186 , 4920 - 4935.e23
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.
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.