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TitleAn AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.
Journal, issue, pagesCell, Vol. 186, Issue 22, Page 4920-4935.e23, Year 2023
Publish dateOct 26, 2023
AuthorsTomohiro Hino / Satoshi N Omura / Ryoya Nakagawa / Tomoki Togashi / Satoru N Takeda / Takafumi Hiramoto / Satoshi Tasaka / Hisato Hirano / Takeshi Tokuyama / Hideki Uosaki / Soh Ishiguro / Madina Kagieva / Hiroyuki Yamano / Yuki Ozaki / Daisuke Motooka / Hideto Mori / Yuhei Kirita / Yoshiaki Kise / Yuzuru Itoh / Satoaki Matoba / Hiroyuki Aburatani / Nozomu Yachie / Tautvydas Karvelis / Virginijus Siksnys / Tsukasa Ohmori / Atsushi Hoshino / Osamu Nureki /
PubMed AbstractSpCas9 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) ...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.
External linksCell / PubMed:37776859
MethodsEM (single particle)
Resolution2.91 - 3.08 Å
Structure data

35912

8j12

EMDB-35912, PDB-8j12:
Cryo-EM structure of the AsCas12f-sgRNA-target DNA ternary complex
Method: EM (single particle) / Resolution: 3.08 Å

35926

8j1j

EMDB-35926, PDB-8j1j:
Cryo-EM structure of the AsCas12f-YHAM-sgRNAS3-5v7-target DNA
Method: EM (single particle) / Resolution: 2.91 Å

35965

8j3r

EMDB-35965, PDB-8j3r:
Cryo-EM structure of the AsCas12f-HKRA-sgRNAS3-5v7-target DNA
Method: EM (single particle) / Resolution: 2.95 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-ZN:
Unknown entry

Source
  • Mycolicibacterium mucogenicum (bacteria)
  • acidibacillus sulfuroxidans (bacteria)
  • acidibacillus sulfooxidans (bacteria)
  • sulfoacidibacillus thermotolerans (bacteria)
KeywordsRNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas / RNA BINDING PROTEIN-DNA COMPLEX / RNA BINDING PROTEIN / RNA BINDING PROTEIN-DNA-RNA complex

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