| Authors |
Hino, Tomohiro ; Omura, Satoshi N ; Nakagawa, Ryoya ; Togashi, Tomoki ; Takeda, Satoru N ; Hiramoto, Takafumi ; Tasaka, Satoshi ; Hirano, Hisato ; Tokuyama, Takeshi ; Uosaki, Hideki ; Ishiguro, Soh ; Kagieva, Madina ; Yamano, Hiroyuki ; Ozaki, Yuki ; Motooka, Daisuke ; Mori, Hideto ; Kirita, Yuhei ; Kise, Yoshiaki ; Itoh, Yuzuru ; Matoba, Satoaki ; Aburatani, Hiroyuki ; Yachie, Nozomu ; Karvelis, Tautvydas ; Šikšnys, Virginijus ; Ohmori, Tsukasa ; Hoshino, Atsushi ; Nureki, Osamu |
| Abstract [eng] |
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. |
