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Title | An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity. |
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Journal, issue, pages | Nat Chem Biol, Vol. 19, Issue 11, Page 1384-1393, Year 2023 |
Publish date | Jul 3, 2023 |
Authors | Tong Wu / Chang Liu / Siyuan Zou / Ruitu Lyu / Bowei Yang / Hao Yan / Minglei Zhao / Weixin Tang / |
PubMed Abstract | 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 ...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 links | Nat Chem Biol / PubMed:37400536 / PubMed Central |
Methods | EM (single particle) |
Resolution | 2.9 Å |
Structure data | EMDB-27801, PDB-8dzj: |
Chemicals | ChemComp-ZN: |
Source |
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Keywords | RNA BINDING PROTEIN/RNA/DNA / CRISPR / Cas12 / gene editing / RNA BINDING PROTEIN-RNA-DNA complex |