Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2026 Oct 13;11(42):64664–64672. doi: 10.1021/acsomega.6c04743

Structure- and Exonuclease-Guided Engineering of Cas12o1 Enhances Genome Editing Efficiency and Deletion Outcomes

Linli Wang ‡,§,∥, Yanlu Wang ‡,§,∥, Xiaoya He ‡,§,∥, Jian Chen †, Yue Ai ‡,§,∥, Hongbing Han †,‡,§,∥,*
PMCID: PMC13717751  PMID: 42959896

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.

graphic file with name ao6c04743_0006.webp

graphic file with name ao6c04743_0004.webp

1. Introduction

Recently identified Cas12o1 is an RNA-guided DNA endonuclease capable of programmable genome editing. Cas12o1 comprises 908 amino acids and is guided by a crRNA consisting of a 36-nt repeat and an 18-nt or longer spacer. Cas12o1 represents an attractive platform for engineering because of several distinctive features. Its compact size facilitates delivery using size-constrained vectors, its distinct ATG protospacer adjacent motif (PAM) expands the repertoire of targetable genomic loci, and the recently resolved cryo-electron microscopy (cryo-EM) structure of the Cas12o1-crRNA-DNA ternary complex provides a structural basis for rational structure-guided engineering. Despite these attractive features, native Cas12o1 exhibits substantially lower editing efficiency in mammalian cells than canonical clustered regularly interspaced short palindromic repeats (CRISPR) nucleases, limiting its practical utility in applications requiring robust on-target activity. Although engineered Cas12o1 variants with improved activity have been reported, several potentially critical mutations remain unexplored.

Notably, Cas12o1 retains cleavage activity with intact spacers extending ≥ 16 bp distal to the PAM, a property that predisposes it to repeated DNA cleavage. In CRISPR-Cas9 systems, such recurrent cutting has been shown to promote high levels of chromosomal translocations, posing a serious threat to genome integrity. T5 exonuclease (T5 exo) is a 5′–3′ exonuclease capable of degrading both single- and double-stranded DNA. Fusion of T5 exo has been demonstrated to enhance genome editing efficiency across multiple CRISPR platforms, including Cas9, Cas12a, Cas12f, and Cas12i. Importantly, T5 exoassisted editing typically induces longer deletionsoften exceeding 10 bp, as observed with Cas12iwhich has been proposed to reduce recurrent cleavage events and potentially mitigate genome instability, although this effect has not been directly evaluated for Cas12o1-based editing.

In this study, we used AlphaFold-based structural modeling to guide a systematic mutational optimization of Cas12o1 and identified a single amino-acid substitution, D534R, that robustly enhances genome editing activity in mammalian cells. To further address recurrent cleavage and DNA repair outcomes, we engineered Cas12o1 through fusion with a 5′–3′ exonuclease, T5 exo, and determined an optimized C-terminal fusion configuration that preserves editing efficiency while strongly biasing repair toward longer deletions. Integration of the D534R mutation with this exonuclease-assisted design yielded an enhanced Cas12o1-T5 exonuclease (enCas12o1-T5 exo) system that consistently improves editing performance across multiple genomic loci. Together, these results establish a highly efficient Cas12o1-based genome editing platform with enhanced capacity to generate long deletions.

2. Results

2.1. Identification of High-Activity Cas12o1 Single-Point Mutants

Guided by the Cas12o1-crRNA-DNA complex structure, we identified 42 solvent-exposed nonarginine residues within the Helical-I/II region that are located near the crRNA-DNA duplex and whose side chains are oriented toward the nucleic acid interface (Figure S1). Because arginine can strengthen electrostatic interactions with the negatively charged DNA phosphate backbone, these positions were selected for systematic arginine-scanning mutagenesis. Expression plasmids encoding each single-site mutant were constructed and transfected into HEK293T cells, and EGFP+ populations were isolated by fluorescence-activated cell sorting (FACS) 48 h post-transfection. Genomic DNA was isolated and the target locus was enriched by nested PCR for high-throughput sequencing and CRISPResso2-based efficiency analysis. This analysis across all 42 mutants identified five key residuesY274R, V281R, S285R, D534R, and T647Rwith Δefficiency increases of 16.61%, 13.46%, 10.79%, 18.13%, and 5.97%, respectively (Figure A–E).

1.

1

Open in a new tab

Identification of the high-activity Cas12o1 mutants through single and combinatorial mutant screening. (A) Strategy for engineering and evaluating Cas12o1 variants. Forty-two single-point mutants were individually generated by PCR-based site-directed mutagenesis, introducing arginine substitutions at predefined positions. Each variant was coexpressed with its crRNA and an EGFP reporter in HEK293T cells, enabling activity-dependent enrichment of EGFP+ cells by fluorescence-activated cell sorting. Editing efficiencies of individual mutants were subsequently quantified by targeted deep sequencing. “R” indicates arginine substitutions. (B–E) Editing efficiencies of selected Cas12o1 single mutants in HEK293T cells, measured by targeted deep sequencing (n = 2 biological replicates; mean ± s.e.m.). These experiments represent an exploratory screening of candidate activity-enhancing mutations. (F) Strategy for engineering and evaluating Cas12o1 variants carrying single or combinatorial mutations. Five high-activity single substitutions (Y274R, V281R, S285R, D534R, and T647R; “R” denotes arginine replacement) were partially combined, expressed in HEK293T cells, and assessed by targeted deep sequencing. (G) Editing efficiencies of selected Cas12o1 mutation combinations in HEK293T cells, quantified by targeted deep sequencing (n = 2 biological replicates; mean ± s.e.m.). (H) Editing activity of Cas12o1 variants containing the D534R substitution at endogenous loci GRIN2B, EMX1 site 1, and EMX1 site 2 in HEK293T cells, measured by targeted deep sequencing. n = 3 biological replicates; mean ± s.e.m., ***p < 0.001; **p < 0.01; ns, not significant (unpaired Student’s t test).

2.2. D534R Emerges as the Most Active Cas12o1 Variant among Single and Combinatorial Mutants

Building on the five activity-enhancing sites identified earlier (Y274R, V281R, S285R, D534R, and T647R), we designed a systematic set of combinatorial mutants (Figure F). Each mutant was individually transfected into HEK293T cells, and EGFP-positive cells were enriched by FACS for targeted amplicon sequencing, followed by CRISPResso2 analysis. All double and triple mutants displayed reduced editing efficiencies compared with the D534R single mutant. Although certain combinations, such as Y274R/V281R, produced slight improvements relative to V281R alone (a 0.93% increase atop its 16.75% enhancement), their activities remained 2.7% lower than that of D534R. These results suggest that once the key catalytic residue D534 is mutated, additional substitutions may introduce steric constraints or negative cooperativity (Figure G). In HEK293T cells, Cas12o1­(D534R) achieved 1.1–2.3-fold the editing efficiencies of Cas12o1 at GRIN2B, EMX1 site 1 and EMX1 site 2 (Figure H). Specifically, the editing efficiencies (indel frequency, %) for Cas12o1 versus Cas12o1­(D534R) were 0.5% vs 1.0% at GRIN2B, 33.8% vs 36.8% at EMX1 site 1, and 46.5% vs 58.0% at EMX1 site 2. Overall, among all single and combinatorial variants, D534R emerged as the most potent enhancer of Cas12o1 editing efficiency.

2.3. T5 Exo Fusion Increases Long-Fragment Deletions by Cas12o1 without Compromising Efficiency

After Cas nucleases generate staggered double-stranded break (DSB) ends, these termini are typically prone to precise repair. In contrast, the presence of T5 exo trims the overhangs, reducing the likelihood of precise end joining and thereby promoting indel formation (Figure A). T5 exo has previously been reported to enhance Cas-mediated editing efficiency and to promote longer deletions. To test whether tethering T5 exo could modulate Cas12o1 repair outcomes, we fused T5 exo to either the N- or C-terminus of Cas12o1 using four linkers(GGGGS)2, (GGGGS)3, SGGSGGSGGS, and XTEN–designated as linker 1 through linker 4, respectively. Here, “N-linker” refers to a fusion configuration in which T5 exo is attached to the N-terminus of Cas12o1 through the indicated linker, whereas “C-linker” refers to T5 exo fused to the C-terminus of Cas12o1 through the corresponding linker. For example, “N-linker 1” denotes T5 exo fused to the N-terminus of Cas12o1 via linker 1 (Figure B). Amplicon sequencing at the FANCF locus, a commonly used site for benchmarking editing efficiency, revealed that the C-linker 1 variant exhibited editing efficiencies comparable to wild-type Cas12o1, whereas all other T5 exo fusions resulted in markedly reduced activity (Figure C). Notably, all T5 exo-Cas12o1 fusions produced a substantially higher proportion of long deletions (≥11 bp), each reaching at least 94.8%, compared with 64.0% for the wild-type Cas12o1 (Figure D–H). Together, these results show that C-terminal fusion of T5 exo via linker 1 preserves editing efficiency while dramatically enriching for long-deletion outcomes.

2.

2

Open in a new tab

T5 exonuclease fusion enhances long-fragment deletions by Cas12o1. (A) Schematic illustrating DNA repair outcomes induced by Cas12o1 with/without T5 exonuclease (T5 exo) fusion. DSB: double-stranded break; indel: insertion/deletion. (B) Design of Cas12o1 fused with T5 exo at either the N- or C-terminus via distinct linkers. (C) Editing efficiencies of Cas12o1 fused with T5 exo at either the N- or C-terminus via different linkers at the FANCF locus in HEK293T cells, quantified by targeted deep sequencing (n = 3 biological replicates; mean ± s.e.m.). “N-linker 1” refers to the fusion of T5 exo to the N-terminal of Cas12o1 using linker 1, with similar meanings for “N-linker 2”, “N-linker 3”, and so on. (D) Cas12o1 fused with T5 exo induces longer genomic deletions at the FANCF locus in HEK293T cells compared with wild-type Cas12o1, as determined by targeted deep sequencing. (E, G) Representative indel size distributions generated by wild-type Cas12o1 and the optimized C-terminal T5 exo fusion variant (C-linker 1) at the FANCF locus in HEK293T cells. (F, H) Top 20 high-frequency on-target mutations profiles at the FANCF locus in HEK293T cells generated by wild-type Cas12o1 and the optimized C-terminal T5 exo fusion variant (C-linker 1), identified by targeted deep sequencing.

2.4. enCas12o1-T5 Exo Exhibits Enhanced Indel Activity and Increased Long-Deletion Frequency

Having identified the most effective amino acid substitutions of Cas12o1 and the advantages conferred by T5 exo fusion, we next sought to integrate these mutations with exonuclease fusion to develop enCas12o1-T5 exo (Figure A). To evaluate the genome-editing capabilities of the optimized variant enCas12o1-T5 exo, we performed targeted deep sequencing at seven representative endogenous target sitesFANCF, EMX1 site 1, EMX1 site 2, CXCR4, GRIN2B, HBB, and CD2in HEK293T cells. On average, enCas12o1-T5 exo generated indels at these seven sites with frequencies of 47.9%. Compared with wild-type Cas12o1, the engineered variant exhibited editing efficiencies ranging from 1.2- to 1.9-fold higher than those of Cas12o1 (Figure B). Notably, at the HBB locus, enCas12o1-T5 exo surpassed SpCas9 and LbCas12a in editing efficiency, while showing comparable or slightly reduced activity at ADRB2, CXCR4, GRIN2B, and CD2 (Figure C).

3.

3

Open in a new tab

Genome-editing activity and deletion profiles of enCas12o1-T5 exo in HEK293T cells. (A) The combination of the Cas12o1 (D534R) variant and T5 exonuclease fusion yields the enhanced Cas12o1-T5 exonuclease (enCas12o1-T5 exo). (B) Indel frequencies generated by wild-type Cas12o1 and enCas12o1-T5 exo at representative endogenous loci (FANCF, EMX1 site 1, EMX1 site 2, CXCR4, GRIN2B, HBB, and CD2) in HEK293T cells, determined by targeted deep sequencing (n = 3 biological replicates; mean ± s.e.m.). (C) Comparison of editing efficiencies among enCas12o1-T5 exo, SpCas9 (Streptococcus pyogenes Cas9) and LbCas12a (Lachnospiraceae bacterium Cas12a) at selected endogenous loci in HEK293T cells, determined by targeted deep sequencing (n = 3 biological replicates; mean ± s.e.m.). (D) Targeted analysis of candidate off-target sites associated with the CXCR4 and GRIN2B targets in HEK293T cells. Indel frequencies were determined by targeted deep sequencing following treatment with wild-type Cas12o1, enCas12o1-T5 exo, or SpCas9. ON, on-target; OT, predicted off-target. (E) Proportion of deletion events exceeding 10 bp generated by wild-type Cas12o1, enCas12o1-T5 exo, SpCas9 and LbCas12a at the indicated endogenous loci in HEK293T cells, determined by targeted deep sequencing. (F) Top 20 high-frequency on-target mutation profiles generated by wild-type Cas12o1, enCas12o1-T5 exo, SpCas9 and LbCas12a at the GRIN2B locus in HEK293T cells, identified by targeted deep sequencing.

To further evaluate nuclease specificity, we predicted candidate off-target sites for the CXCR4 and GRIN2B targets and quantified editing at these sites by targeted deep sequencing. Both wild-type Cas12o1 and enCas12o1-T5 exo displayed minimal indel formation across the candidate sites, with no appreciable increase in off-target activity for enCas12o1-T5 exo. By contrast, SpCas9 exhibited detectable editing at one predicted off-target site (Figure D). These results indicate that the enhanced editing activity of enCas12o1-T5 exo is achieved without an apparent compromise in target specificity.

To assess the impact of enCas12o1-T5 exo on deletion length, we performed deep sequencing analysis of PCR amplicons encompassing the gRNA target sites in HEK293T cells. The enCas12o1-T5 exo system induced a higher proportion of deletions greater than 10 bp at GRIN2B, CXCR4, and HBB, whereas wild-type Cas12o1, SpCas9, and LbCas12a generated a substantially lower proportion of deletions exceeding 10 bp (Figure E,F). These findings indicate that enCas12o1-T5 exo favors the generation of larger deletions.

3. Discussion

We systematically screened 42 single-amino acid substitutions in Cas12o1 and identified D534R as the most effective variant, increasing editing efficiency by 1.1–2.3-fold. Although several additional mutations (e.g., Y274R, V281R, S285R and T647R) exhibited moderate activity improvements in the initial screen, these observations should be regarded as preliminary because they were obtained from exploratory screening experiments and require further experimental validation. In parallel, we evaluated eight T5 exo fusion architectures and found that C-terminal fusion via a (GGGGS)2 linker was optimal, elevating the proportion of deletions >10 bp from 64.0% to 97.0% without compromising editing efficiency. Combining D534R with this fusion further enhanced activity by 1.2–1.9-fold across seven endogenous target sites, including FANCF, EMX1, and CXCR4, yielding an engineered Cas12o1 variant with improved potency and a strongly biased long-deletion profile. Although SpCas9 and LbCas12a were included as benchmarks, direct comparisons among different nucleases should be interpreted with caution because their distinct PAM requirements necessitate different guide sequences.

Structural analysis based on the reported cryo-EM structure of the Cas12o1-crRNA-DNA ternary complex suggests that residue D534 is located in close proximity to the crRNA-binding interface and the RuvC catalytic region (Figure S2). This spatial arrangement is consistent with the possibility that replacing the negatively charged aspartate with the positively charged arginine could enhance local electrostatic interactions with the crRNA and/or target DNA, potentially stabilizing the ribonucleoprotein–DNA complex and thereby contributing to the enhanced editing activity observed in this study. Its proximity to the RuvC catalytic region further suggests that the D534R substitution may influence local conformational dynamics during target recognition or DNA cleavage. However, these interpretations are based on structural analysis of the reported cryo-EM structure and should therefore be regarded as mechanistic hypotheses rather than experimentally validated conclusions. As the primary objective of this study was to engineer Cas12o1 variants with improved genome-editing performance, elucidating the precise molecular mechanism underlying the D534R substitution was beyond the scope of the present work and warrants future structural and biochemical investigation. Notably, combinatorial mutations reduced editing efficiency (Figure G), suggesting that additional substitutions may perturb local structural organization rather than further improving Cas12o1 activity. T5 exo has previously been shown to enhance editing efficiency and promote longer deletions when fused to Cas nucleases such as Cas9, Cas12a, and Cas12i. ,− Consistent with these observations, T5 exo fusion in our system markedly increased the frequency of long deletions, although its effect on overall Cas12o1 editing efficiency was modest.

The ability to generate extended deletions is particularly valuable for targeting cis-regulatory elementssuch as transcription factor binding sites, enhancers, silencers, and miRNA-binding regionswhich typically span 10–40 bp. Inducing defined long deletions at these loci has become an effective strategy for generating quantitative trait variation and allelic diversity in crops, including rice, soybean, tomato, and maize. However, the biological consequences of long deletions may depend on the specific application context. While extended deletions can facilitate the disruption of regulatory regions and the generation of defined functional alleles, their potential to introduce unintended genomic alterations should also be carefully evaluated, particularly in therapeutic applications requiring precise genome modification. Conventional approaches often rely on multiplexed guide RNAs to achieve large deletions; , however, this strategy is labor-intensive and may elevate off-target risk. Repetitive cleavage events, especially in CRISPR-Cas9 systems, can also increase chromosomal translocations, posing threats to genome integrity. The engineered enCas12o1-T5 exo platform described here provides a streamlined approach for generating long deletions using a single guide, although whether this strategy reduces recurrent cleavage-associated genomic instability remains to be experimentally determined. Nevertheless, our evaluation of enCas12o1-T5 exo was limited to HEK293T cells, which provide a well-established and widely used platform for evaluating engineered CRISPR systems under standardized experimental conditions. Because genome editing efficiency and DNA repair outcomes can vary across different cellular contexts owing to differences in chromatin accessibility, DNA repair pathways, and delivery efficiency, further validation in additional mammalian cell types, primary cells, and in vivo models will be important to define the broader applicability of enCas12o1-T5 exo. Similarly, the present mutational screen was restricted to residues within the Helical-I/II region; therefore, additional activity-enhancing mutations in other structural domains of Cas12o1 may remain to be identified. Although the engineered Cas12o1 variants showed improved genome-editing performance, we cannot completely exclude the possibility that altered protein expression or intracellular stability may contribute to the observed effects. In addition to on-target activity, we assessed potential off-target effects of Cas12o1 and enCas12o1-T5 exo by targeted deep sequencing of predicted candidate sites for CXCR4 and GRIN2B. Both variants showed minimal indel formation, with no appreciable increase in off-target activity following T5 exo fusion. Nevertheless, targeted deep sequencing of predicted sites provides a focused assessment rather than a comprehensive evaluation of genome-wide specificity. Because enhanced nuclease activity may potentially alter editing specificity, future studies employing unbiased genome-wide approaches, such as GUIDE-seq, , DISCOVER-seq, , CIRCLE-seq or related methods, will be required to comprehensively evaluate the specificity and safety profile of enCas12o1-T5 exo before broader applications.

In summary, through systematic amino acid mutagenesis and optimized T5 exo fusion, we developed enCas12o1-T5 exo, a Cas12o1-derived editor with enhanced activity and a pronounced capacity to generate long deletions. These features position it as a promising platform for applications ranging from functional genomics to agricultural biotechnology.

4. Methods

4.1. Plasmid Construction

Mammalian codon-optimized wild-type Cas12o1 fused to C-Myc, BP-SV40, and nucleoplasmin nuclear localization signals, together with the linker and T5 exo, was synthesized by GeneWiz Co., Ltd. These elements were assembled into a CBh-driven mammalian expression vector generated by AgeI/FseI digestion of pX458 (Addgene #48138) using seamless cloning. Cas12o1 mutants were produced by PCR amplification of two Cas12o1 fragments from the above wild-type template using mutation-bearing primers, followed by vector construction as described. Short DNA oligos encoding crRNA were also synthesized by GeneWiz Co., Ltd., amplified by PCR, and cloned into the same expression vector via BbsI/KpnI sites. The resulting plasmids contain a human U6 promoter to drive crRNA expression. All sequences are provided in Table S1.

4.2. Cell Culture and Transfection

HEK293T cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco) and 1% (v/v) penicillin-streptomycin (100×; Gibco) at 37 °C in a 5% CO2 incubator. Upon reaching ∼90% confluence, cells were dissociated and seeded into 24-well plates (Corning). When reaching 80–90% confluence the next day, cells were transfected with 500 ng plasmid DNA using Lipofectamine 3000 (Thermo Fisher Scientific) following the manufacturer’s protocol.

4.3. Evaluation of on-Target and off-Target Editing

Forty thousand EGFP-positive cells were isolated 48 h post-transfection using a BD FACSAria III. Genomic DNA was extracted with the HiPure Tissue DNA Micro Kit (Magen), and target loci were amplified using PrimeSTAR Max DNA Polymerase (Takara) with primers listed in Tables S2–S4. PCR products were purified using the TaKaRa MiniBEST DNA Fragment Purification Kit v4.0 (Takara) and subsequently sequenced on a DNBSEQ-T7 platform (150-bp paired-end; Benagen Co., Ltd.). Sequencing reads were analyzed with CRISPResso2 to determine editing efficiencies. For off-target analysis, candidate off-target sites associated with the CXCR4 and GRIN2B targets were computationally predicted and subsequently amplified and analyzed by targeted deep sequencing using the same workflow. The candidate off-target sites and corresponding primer sequences are provided in Tables S5 and S6.

4.4. Statistical Analysis

All quantitative data are presented as mean ± standard error of the mean (s.e.m.). Sample sizes (n) represent independent biological replicates, and no technical replicates were included in statistical analyses. Statistical significance between two groups was assessed using a two-tailed unpaired Student’s t test. Because the sample sizes were limited (n = 2 or n = 3), formal normality testing was not performed, as such tests have insufficient statistical power to reliably assess data distribution with small sample sizes. Instead, the use of parametric testing was based on the assumption that the measured biological variables approximately followed a normal distribution. Differences were considered statistically significant at P < 0.05. The initial mutagenesis screening (Figures B–G) was performed using two independent biological replicates as an exploratory screen to identify candidate variants for subsequent validation. All major conclusions were subsequently validated using three independent biological replicates.

Supplementary Material

ao6c04743_si_001.pdf (538.1KB, pdf)

Acknowledgments

We thank the Flow Cytometry Facility (Mr. Yunlong Wang, Ms. Chunguang Han, and Ms. Yanan Hao) of the National Center for Protein Sciences Beijing (NCPSB) for their assistance.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c04743.

  • Spatial distribution of the 42 mutations in Cas12o1 (Figure S1); structure model of the Cas12o1­(D534R)–crRNA–dsDNA ternary complex (Figure S2); protein and coding sequences, linker sequences, guide sequences, and PCR primers used in this study (Tables S1–S4); guide sequences used for on-target and predicted off-target analysis of CXCR4 and GRIN2B (Table S5); and PCR primers used for targeted deep sequencing of on-target and predicted off-target sites (Table S6) (PDF)

⊥.

L.W., Y.W., and X.H. contributed equally to this work. L.W. drafted the initial manuscript; L.W., Y.W., and X.H. performed the experiments; L.W. and Y.W. analyzed the targeted deep sequencing data; J.C. and Y.A. provided feedback on the manuscript; H.H. conceived the project, supervised and designed all research, and revised the manuscript.

This study was supported by the Projects of Unveiling List and Entrusting the Leader for Science and Technology Breakthrough in Inner Mongolia (2025KJTW0022), the Chinese Universities Scientific Fund (2026TC112 and 2022TC025), the Great Projects of Agricultural Biotechnology Breeding in China (2022ZD04013), and the Pinduoduo-China Agricultural University Research Fund (PC2023A01004).

The authors declare no competing financial interest.

References

  1. Duan Z., Zhang X., Zhang J. T., Ji X., Liu R., Chen Y.. et al. Structure and genome editing activity of the novel CRISPR-Cas12o1 effector. Cell Res. 2025;35(2):145–148. doi: 10.1038/s41422-024-01050-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Yin J., Lu R., Xin C., Wang Y., Ling X., Li D.. et al. Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing. Nat. Commun. 2022;13(1):1204. doi: 10.1038/s41467-022-28900-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Zhang Q., Yin K., Liu G., Li S., Li M., Qiu J. L.. Fusing T5 exonuclease with Cas9 and Cas12a increases the frequency and size of deletion at target sites. Sci. China Life Sci. 2020;63(12):1918–1927. doi: 10.1007/s11427-020-1671-6. [DOI] [PubMed] [Google Scholar]
  4. Liao K., Chen K., Ma S., Yang X., Chen P., Li S.. et al. exoCasMINI: A T5 exonuclease fused CRISPR-Cas12f system with enhanced gene editing efficiency. iScience. 2025;28(8):113171. doi: 10.1016/j.isci.2025.113171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Wang L., Wang Y., Chen J., Zhu Y., Qin H., Liu J.. et al. An engineered CRISPR-Cas12i tool for efficient multiplexed genome editing. Nucleic Acids Res. 2025;53(16):gkaf806. doi: 10.1093/nar/gkaf806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Liang Z., Wu Y., Guo Y., Wei S.. Addition of the T5 exonuclease increases the prime editing efficiency in plants. J. Genet. Genomics. 2023;50(8):582–588. doi: 10.1016/j.jgg.2023.03.008. [DOI] [PubMed] [Google Scholar]
  7. Wu Y., Yuan Q., Zhu Y., Gao X., Song J., Yin Z.. Improving FnCas12a genome editing by exonuclease fusion. CRISPR J. 2020;3(6):503–511. doi: 10.1089/crispr.2020.0073. [DOI] [PubMed] [Google Scholar]
  8. Tang J., Chu C.. MicroRNAs in crop improvement: fine-tuners for complex traits. Nat. Plants. 2017;3:17077. doi: 10.1038/nplants.2017.77. [DOI] [PubMed] [Google Scholar]
  9. Wang L., O’Conner S., Tanvir R., Zheng W., Cothron S., Towery K.. et al. CRISPR/Cas9-based editing of NF-YC4 promoters yields high-protein rice and soybean. New Phytol. 2025;245(5):2103–2116. doi: 10.1111/nph.20141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Rodríguez-Leal D., Lemmon Z. H., Man J., Bartlett M. E., Lippman Z. B.. Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing. Cell. 2017;171(2):470–480.e8. doi: 10.1016/j.cell.2017.08.030. [DOI] [PubMed] [Google Scholar]
  11. Liu L., Gallagher J., Arevalo E. D., Chen R., Skopelitis T., Wu Q.. et al. Enhancing grain-yield-related traits by CRISPR-Cas9 promoter editing of maize CLE genes. Nat. Plants. 2021;7(3):287–294. doi: 10.1038/s41477-021-00858-5. [DOI] [PubMed] [Google Scholar]
  12. Malinin N. L., Lee G., Lazzarotto C. R., Li Y., Zheng Z., Nguyen N. T.. et al. Defining genome-wide CRISPR-Cas genome-editing nuclease activity with GUIDE-seq. Nat. Protoc. 2021;16(12):5592–5615. doi: 10.1038/s41596-021-00626-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Tsai S. Q., Zheng Z., Nguyen N. T., Liebers M., Topkar V. V., Thapar V.. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 2015;33(2):187–197. doi: 10.1038/nbt.3117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Wienert B., Wyman S. K., Yeh C. D., Conklin B. R., Corn J. E.. CRISPR off-target detection with DISCOVER-seq. Nat. Protoc. 2020;15(5):1775–1799. doi: 10.1038/s41596-020-0309-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Wienert B., Wyman S. K., Richardson C. D., Yeh C. D., Akcakaya P., Porritt M. J.. et al. Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. Science. 2019;364(6437):286–289. doi: 10.1126/science.aav9023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Tsai S. Q., Nguyen N. T., Malagon-Lopez J., Topkar V. V., Aryee M. J., Joung J. K.. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat. Methods. 2017;14(6):607–614. doi: 10.1038/nmeth.4278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Clement K., Rees H., Canver M. C., Gehrke J. M., Farouni R., Hsu J. Y.. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 2019;37(3):224–226. doi: 10.1038/s41587-019-0032-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao6c04743_si_001.pdf (538.1KB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES