An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.
Cell
; 186(22): 4920-4935.e23, 2023 10 26.
Article
em En
| MEDLINE
| ID: mdl-37776859
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.
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Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Sistemas CRISPR-Cas
/
Edição de Genes
Limite:
Animals
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Humans
Idioma:
En
Revista:
Cell
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Japão