Structure- and Exonuclease-Guided Engineering of Cas12o1 Enhances Genome Editing Efficiency and Deletion Outcomes
- 1China Agricultural University , , ,
Abstract
Small and efficient CRISPR nucleases hold great promise for genome engineering but often exhibit suboptimal activity and limited control over DNA repair outcomes. Here, we engineer Cas12o1 using a structure- and exonuclease-guided strategy to enhance both editing efficiency and deletion profiles in mammalian cells. Structure-guided arginine-scanning mutagenesis of 42 residues identifies a single substitution, D534R, that markedly improves editing efficiency across multiple genomic loci, outperforming combinatorial variants. In parallel, fusion of T5 exonuclease (T5 exo) profoundly remodels DNA repair outcomes, strongly enriching long-fragment deletions without compromising overall activity. Integrating the D534R mutation with T5 exo fusion increases Cas12o1 editing efficiency by 1.2–1.9-fold and yields a higher proportion of deletions exceeding 10 bp. Notably, at selected loci such as HBB, the enhanced Cas12o1-T5 exonuclease (enCas12o1-T5 exo) achieved comparable or higher editing efficiencies than canonical SpCas9 and LbCas12a. Together, these results establish a rational framework for simultaneously enhancing the efficiency and repair outcomes of compact CRISPR nucleases, expanding the genome-engineering potential of Cas12o1.
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BibTeX
@article{wang2026structure,
title = {{Structure- and Exonuclease-Guided Engineering of Cas12o1 Enhances Genome Editing Efficiency and Deletion Outcomes}},
author = {Linli Wang and Yanlu Wang and Xiaoya He and Jian Chen and Yue Ai and Hongbing Han},
journal = {ACS Omega},
year = {2026},
month = oct,
doi = {10.1021/acsomega.6c04743},
url = {https://pubs.acs.org/acsodf/article/11/42/64664/5449626/Structure-and-Exonuclease-Guided-Engineering-of},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Structure- and Exonuclease-Guided Engineering of Cas12o1 Enhances Genome Editing Efficiency and Deletion Outcomes
AU - Linli Wang
AU - Yanlu Wang
AU - Xiaoya He
AU - Jian Chen
AU - Yue Ai
AU - Hongbing Han
PY - 2026
DA - 2026/10/13
JO - ACS Omega
DO - 10.1021/acsomega.6c04743
UR - https://pubs.acs.org/acsodf/article/11/42/64664/5449626/Structure-and-Exonuclease-Guided-Engineering-of
AB - Small and efficient CRISPR nucleases hold great promise for genome engineering but often exhibit suboptimal activity and limited control over DNA repair outcomes. Here, we engineer Cas12o1 using a structure- and exonuclease-guided strategy to enhance both editing efficiency and deletion profiles in mammalian cells. Structure-guided arginine-scanning mutagenesis of 42 residues identifies a single substitution, D534R, that markedly improves editing efficiency across multiple genomic loci, outperforming combinatorial variants. In parallel, fusion of T5 exonuclease (T5 exo) profoundly remodels DNA repair outcomes, strongly enriching long-fragment deletions without compromising overall activity. Integrating the D534R mutation with T5 exo fusion increases Cas12o1 editing efficiency by 1.2–1.9-fold and yields a higher proportion of deletions exceeding 10 bp. Notably, at selected loci such as HBB, the enhanced Cas12o1-T5 exonuclease (enCas12o1-T5 exo) achieved comparable or higher editing efficiencies than canonical SpCas9 and LbCas12a. Together, these results establish a rational framework for simultaneously enhancing the efficiency and repair outcomes of compact CRISPR nucleases, expanding the genome-engineering potential of Cas12o1.
SN - 2470-1343
ER -