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TitleAn engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity.
Journal, issue, pagesNat Chem Biol, Vol. 19, Issue 11, Page 1384-1393, Year 2023
Publish dateJul 3, 2023
AuthorsTong Wu / Chang Liu / Siyuan Zou / Ruitu Lyu / Bowei Yang / Hao Yan / Minglei Zhao / Weixin Tang /
PubMed AbstractCompact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered ...Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.
External linksNat Chem Biol / PubMed:37400536 / PubMed Central
MethodsEM (single particle)
Resolution2.9 Å
Structure data

EMDB-27801, PDB-8dzj:
Cryo-EM structure of Acidibacillus sulfuroxidans Cas12f in complex with sgRNA and target DNA
Method: EM (single particle) / Resolution: 2.9 Å

Chemicals

ChemComp-ZN:
Unknown entry

Source
  • acidibacillus sulfuroxidans (bacteria)
  • homo sapiens (human)
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR / Cas12 / gene editing / RNA BINDING PROTEIN-RNA-DNA complex

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