AlphaFold3-guided tracrRNA redesign yields small monomeric Cas12f RNPs

Lulu Pan; Rui Sang; Ruier Xue; Yongcheng Ma; Ewa Goldys; Fei Deng

doi:10.1093/nar/gkag430

. 2026 May 4;54(8):gkag430. doi: 10.1093/nar/gkag430

AlphaFold3-guided tracrRNA redesign yields small monomeric Cas12f RNPs

Lulu Pan ^1,^2,^3,⁴, Rui Sang ^5,⁶, Ruier Xue ^7,⁸, Yongcheng Ma ^9,¹⁰, Ewa Goldys ^11,^12,^✉, Fei Deng ^13,^14,^✉

¹ School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia

² ARC Centre of Excellence for Nanoscale Biophotonics, University of New South Wales, Sydney 2052, Australia

³ Central China Fuwai Hospital of Zhengzhou University, Henan Provincial People’s Hospital, Zhengzhou, Henan 450003, China

⁴ Henan Key Laboratory of Individualized Drug Therapy for Cardiovascular Diseases, Zhengzhou, Henan 450003, China

⁵ School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia

⁶ ARC Centre of Excellence for Nanoscale Biophotonics, University of New South Wales, Sydney 2052, Australia

⁷ School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia

⁸ ARC Centre of Excellence for Nanoscale Biophotonics, University of New South Wales, Sydney 2052, Australia

⁹ Central China Fuwai Hospital of Zhengzhou University, Henan Provincial People’s Hospital, Zhengzhou, Henan 450003, China

¹⁰ Henan Key Laboratory of Individualized Drug Therapy for Cardiovascular Diseases, Zhengzhou, Henan 450003, China

¹¹ School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia

¹² ARC Centre of Excellence for Nanoscale Biophotonics, University of New South Wales, Sydney 2052, Australia

¹³ School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia

¹⁴ ARC Centre of Excellence for Nanoscale Biophotonics, University of New South Wales, Sydney 2052, Australia

^✉

To whom correspondence should be addressed. Email: e.goldys@unsw.edu.au

^✉

Correspondence may also be addressed to Fei Deng. Email: fei.deng@unsw.edu.au

Roles

Lulu Pan: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft

Rui Sang: Data curation, Formal analysis, Investigation, Writing - review & editing

Ruier Xue: Data curation, Investigation

Yongcheng Ma: Writing - review & editing

Ewa Goldys: Supervision, Writing - review & editing

Fei Deng: Conceptualization, Funding acquisition, Project administration, Supervision, Writing - review & editing

PMCID: PMC13137049 PMID: 42080267

Abstract

Although Cas12f (Cas14) is among the smallest Class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effectors, it assembles into dimeric ribonucleoprotein (RNP) complexes with guide RNA, substantially increasing its functional size and limiting its suitability for gene editing and biosensing applications. To overcome this limitation, we systematically investigate the structural and functional roles of Cas12f dimerization using a combination of computational modeling and experimental validation. Structural analysis using Protein Data Bank data and AlphaFold-3 predictions revealed that the 5′-end sequence of tracrRNA is essential for dimer formation but dispensable for substrate cleavage. Based on this, we designed a truncated tracrRNA by removing 70 nucleotides from its 5′-end. This shortened tracrRNA successfully loaded into Cas12f to form a one guide RNA–one Cas12f monomer RNP. This functionally monomeric RNP demonstrated substantially enhanced trans-cleavage activity: 4.5-fold for ssDNA, 3.5-fold for dsDNA, and 2.5-fold for RNA, resulting in markedly improved detection sensitivity: 10-fold for ssDNA and dsDNA, and 4-fold for RNA. In addition, the functionally monomeric RNP exhibits cis-cleavage activity and gene editing efficiency comparable to that of the dimeric RNP, thereby restoring the advantage of Cas12f as a compact enzyme for in vivo gene editing. These results highlight that the functionally monomeric Cas12f RNP combines enhanced biosensing performance with retention of its uniquely compact size, benefiting gene editing applications.

Graphical Abstract

For image description, please refer to the figure legend and surrounding text.

Introduction

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are pivotal tools in modern biotechnology, enabling gene editing and nucleic acid detection [1, 2]. CRISPR-based technologies primarily rely on Class 2 CRISPR RNA-guided enzymes, such as Cas9 [3, 4], Cas12 [5–9], and Cas13 [10–13]. These systems, programmed by CRISPR RNA (crRNA), specifically recognize target strands containing sequences homologous to the crRNA either in a protospacer adjacent motif (PAM)-dependent or in an independent manner. This recognition activates the Cas proteins’ sequence-specific cis-cleavage, as well as their nonspecific trans-cleavage activity.

Cas12f, also known as Cas14, is a unique subtype of Cas12. Unlike other Cas enzymes, Cas12f is ∼400–700 amino acids in length, making it about half the size of other Class 2 CRISPR enzymes, which are typically 950–1400 amino acids [14]. Cas12f’s target recognition and activation of cis- and trans-cleavage activities are mediated by a complex of trans-activating CRISPR RNA (tracrRNA) and crRNA. Structural studies have revealed that Cas12f forms a dimeric complex with one gRNA (one tracrRNA and one crRNA), but only one of the two Cas12f subunits exhibits trans-cleavage activity. This implies that the second subunit is functionally redundant [15, 16], resulting in suboptimal utilization of the trans-cleavage function and potentially limiting its performance in biosensing and/or gene editing applications. Moreover, the dimeric configuration substantially increases the size of the ribonucleoprotein (RNP) complex, posing challenges for in vivo gene editing applications. Therefore, engineering Cas12f to function as a monomer is crucial for enhancing its utility in both gene editing and biosensing contexts.

In this study, we aimed to eliminate the Cas12f RNP dimerization by first analyzing its structural basis using Protein Data Bank (PDB) data and AlphaFold 3 (AF3) predictions [17–19]. Our analysis indicates that the 5′-end sequence of tracrRNA mediates dimer formation via the noncleaving Cas12f.2 subunit, while not being required for cleavage activity. Based on this, we hypothesize that removing the first 70 nucleotides of the tracrRNA will not affect Cas12f-mediated cleavage. Accordingly, we designed a truncated tracrRNA by reducing the 5′-end sequence by 70 nucleotides. This truncated tracrRNA produces a functionally monomeric Cas12f RNP, which maintains comparable cis-cleavage activity while exhibiting enhanced trans-cleavage activity.

Materials and methods

Chemicals and biological reagents

Cas12f (Magigen, C015M), NEB1, NEB2.1, NEB3, NEB4, and rCutSmart buffers (New England Biolab), agarose (Thermo Fisher), TAE buffer (Bio-Rad), SYBR Gold DNA dye (Thermo Fisher), 10 bp DNA ladder (Thermo Fisher), 6X DNA loading dye (Thermo Fisher), DNase/RNase-free water (Thermo Fisher), NativePAGE 4%–16% Bis-Tris Protein Gels (Thermo Fisher), NativePAGE Sample Buffer (Thermo Fisher), NativePAGE Running Buffer Kit (Thermo Fisher), and PageRuler Prestained Protein Ladder (Thermo Fisher), Opti-MEM™ I Reduced Serum Medium (Gibco, Thermo Fisher Scientific), Lipofectamine™ Cas9 Plus Reagent (Invitrogen, Thermo Fisher Scientific), and CRISPRMAX™ Transfection Reagent (Invitrogen, Thermo Fisher Scientific) were used in the study. All DNA and RNA oligos were synthesized by Gencefe, and sequence information is provided in Supplementary Table S1.

Structural simulation of Cas12f RNP

The structure of Cas12f RNP was simulated using the AF3 server (https://alphafoldserver.com/). The specific sequences used in simulations are listed in Supplementary Table S2. The protein, guide RNA, and DNA trigger sequences were input into separate columns, with a single copy of each molecule used for simulations. Then, the job was submitted with Auto seed, and multiple independent runs produced consistent structural features. The resulting structures were visualized using Visual Studio Code (VS Code, Version 1.91.1). The simulation profiles (model-0.cif) generated by AF3 were analyzed, and key amino acids were highlighted for detailed analysis.

Structural comparison of different complexes

The global structures of different complexes were compared using the PyMOL Molecular Graphics System (Version 2.6 Schrödinger, LLC). Protein profiles (.cif) from PDB or AF3 were loaded into PyMOL, and the root mean square deviation (RMSD) was calculated to assess structural differences between complexes. Significant structural differences were indicated by an RMSD value >2 Å [20]. The distance between the residues Val377 and Arg490 in Cas12f RNPs was also measured to assess the degree of opening in the active pocket [15].

Evaluating trans-cleavage activity of Cas12f RNP with truncated and full-length tracrRNA

The trans-cleavage activity of Cas12f RNP with truncated tracrRNA and Cas12f RNP with full-length tracrRNA was compared using a variety of target nucleic acids. The reaction mixture was prepared by mixing 6 µl (for truncated tracrRNA) or 12 µl (for full-length tracrRNA) of 5 µM Cas12f, 3 µl of 10 µM tracrRNA, 3 µl of 10 µM crRNA, and 6 µl of 100 µM Texas Red-18T-BHQ2 reporter in 600 µl of 1× rCutSmart buffer. Cas12f was activated by adding 5 µl of 1 µM target/water to 95 µl of the prepared reaction mixture. Fluorescence was measured using an ID3 plate reader with excitation/emission wavelengths of 570 nm/615 nm at 46°C for 1 h.

Limit of detection test for Cas12f RNP with truncated tracrRNA and full-length tracrRNA

For limit of detection (LOD) test, a reaction mixture was prepared by mixing 18 µl (for truncated tracrRNA) or 36 µl (for full-length tracrRNA) of 5 µM Cas12f, 9 µl of 10 µM of tracrRNA and crRNA, and 18 µl of 100 µM Texas Red-18T-BHQ2 reporter in 1800 µl of 1× rCutSmart buffer. Cas12f was activated by adding 5 µl of various concentrations of target (10 nM, 1 nM, 100 pM, 10 pM, or 1 pM) or water to 95 µl of the prepared reaction mixture. Fluorescence was recorded using an ID3 plate reader with excitation/emission wavelengths of 570 nm/615 nm at 46°C for 1 h.

Specificity test for Cas12f RNP with truncated tracrRNA and full-length tracrRNA

For specificity test, a standard reaction mixture was prepared by mixing 15 µl (for truncated tracrRNA) or 30 µl (for full-length tracrRNA) of 5 µM Cas12f, 7.5 µl of 10 µM of tracrRNA and crRNA, and 15 µl of 100 µM Texas Red-18T-BHQ2 reporter in 1500 µl of 1× rCutSmart buffer. Cas12f was activated by adding 5 µl of 1 µM target with different single nucleotide polymorphisms (SNPs) or water to 95 µl of the prepared reaction mixture. Fluorescence was recorded using an ID3 plate reader with excitation/emission wavelengths of 570 nm/615 nm at 46°C for 1 h.

In vitro transfection efficiency measurement

To further verify the cis-cleavage activity of Cas12f RNP, we evaluated the in vitro transfection efficiency for GFP gene knockout using the Lipofectamine CRISPRMAX delivery system. MDA-MB-231-GFP cells were seeded into six-well plates or glass-bottom Petri dishes at a density of 3 × 10⁵ cells per well and cultured for 24 h. Transfection was performed according to the manufacturer’s protocol (Invitrogen TrueGuide™ Synthetic gRNA, Thermo Fisher Scientific, Waltham, MA, USA). Specially, 125 μl of Opti-MEM™ I Reduced Serum Medium was mixed with 37.5 pmol of Cas12f protein, 37.5 pmol of gRNA, and 12.5 μl of Lipofectamine™ Plus Reagent in one tube (Tube 1). Separately, 125 μl of Opti-MEM™ I Medium was combined with 7.5 μl of CRISPRMAX™ Transfection Reagent in another tube (Tube 2) and incubated at room temperature for 1 min. The contents of Tube 2 were then added to Tube 1, and the mixture was incubated at room temperature for 10 min to facilitate CRISPR–Cas12f RNP complex formation. Following incubation, 250 μl of the transfection complex was added to each well, and cells were incubated at 37°C with 5% CO₂. Transfection efficiency was assessed 72 h post-transfection by flow cytometry (BD LSRFortessa™ SORP, BD Biosciences, San Jose, CA, USA) and confocal laser scanning microscopy (Olympus FV3000). The percentage of GFP-positive cells was quantified for comparison between experimental conditions. Flow cytometry analysis was performed with FlowJo v10.10.0 software. GFP fluorescence was detected using an excitation wavelength of 488 nm and an emission wavelength of 530 nm for flow cytometry, while confocal imaging was conducted with an emission detection range of 500–550 nm.

All the gene technology research was approved by UNSW Gene Technology Research Committee (iRECS9068).

Statistical analyses

For optimization of reaction conditions, t-test, one-way Analysis of Variance (ANOVA), or two-way ANOVA was utilized based on conditions. In the ANOVA test, Bonferroni correction was adopted to control false discovery rate in multiple comparisons. For trans-cleavage activity comparisons for Cas12f RNP with full-length and truncated tracrRNA, as well as different ratios of Cas:gRNA, two-way ANOVA was used to analyze the significance of difference. For sensitivity, LOD was determined by signal to background (S/B) > 3. For specificity, one-way ANOVA was used to obtain P-value when compared with perfect match (PM) sequence.

Results

Structure simulation of Cas12f with truncated and full-length tracrRNA

Given that the active sites of Cas12f—including D326, E422, and D510 from RuvC, along with R490 from Nuc—are occluded by the lid motif (L424-F442) in the absence of target DNA and become accessible in its presence [15], we analyzed structural changes induced by target binding. Specifically, we measured the distance between R490 in the active site and S433 in the lid to assess the opening of the active pocket and its impact on cleavage activity, because R490 in the active site and S433 in the lid are both at the edge of the catalytic pocket. As expected, the distance in Cas12f.1 increased from 10.15 Å (7L48, inactive Cas12f RNP) to 17.11 Å (7L49, active Cas12f RNP) upon the presence of target DNA, while Cas12f.2 showed no significant change (Supplementary Fig. S1). This aligns with the finding that Cas12f.1, rather than Cas12f.2, is responsible for substrate cleavage [15]. Structural analysis indicates that the 5′-end sequence of tracrRNA binds predominantly to Cas12f.2, which is not involved in substrate cleavage (Supplementary Fig. S2). Based on this finding, we hypothesized that this sequence is necessary for dimer formation but not for cleavage activity. To test this hypothesis, AF3 was used to simulate the structure of activated Cas12f RNP with monomeric Cas protein, revealing that the 5′-end sequence of tracrRNA indeed does not interact with the Cas12f protein (Fig. 1A, left). The deletion of sequences from C1 to U70 generated a 5′-end truncated tracrRNA, which was capable of forming a complex with the intact Cas12f RNP (Fig. 1A, right). The two Cas12f complexes exhibited significant structural differences, with a high RMSD of 2.422 Å (Fig. 1B). This raises the question of whether Cas12f RNP with the truncated tracrRNA retains its substrate cleavage function.

Truncation of tracrRNA converts the Cas12f RNP from a dimetric to a functionally monomeric form

Based on the deletion of the 5′-end sequence of tracrRNA, we hypothesized that only one Cas12f protein associates with the truncated tracrRNA. To verify this hypothesis, different ratios of Cas and gRNA were assessed, which showed that the trans-cleavage activity for the Cas:gRNA (full-length) ratio of 2:1 had the highest trans-cleavage activity (Fig. 2A), as previously reported, whereas Cas:gRNA (truncated) ratio of 1:1 showed the highest trans-cleavage activity compared with other ratios (Fig. 2B). Furthermore, AF3 predictions suggested that upon binding target DNA, Cas12f monomer RNP may adopt an activated conformation, as indicated by the increase in distance between Ser433 and Arg490 from 14.25 to 16.26 Å (Fig. 2C and D). Based on this structural hypothesis, we will evaluate the activity of functionally monomeric Cas12f RNP using sensitivity and specificity assays with different DNA targets, which can support the possibility that target DNA can activate the functionally monomeric Cas12f RNP.

Comparison of Cas12f functionally monomeric RNP with dimeric RNP on biosensing applications

To determine the optimal reaction conditions for Cas12f RNP, we systematically evaluated the basic trans-cleavage reaction parameters, and found that the optimal buffer was rCutSmart, optimal temperature was 46°C, and reporter was 18T ssDNA (Supplementary Fig. S3). Under the optimal reaction conditions, the trans-cleavage activities of Cas12f RNP with truncated (monomer) and full-length tracrRNA (dimer) were compared. The results show that Cas12f functionally monomeric RNP exhibits superior trans-cleavage activity compared to dimeric RNA, regardless of whether it was triggered by target ssDNA (4.5 times) (Fig. 3A), RNA (2.5 times) (Fig. 3B), or dsDNA (with or without PAM) (2.6–3.5 times) (Fig. 3C and D).

Furthermore, the sensitivity and specificity of Cas12f monomer RNP activated by different targets were explored. For the sensitivity, Cas12f functionally monomeric RNP had the sensitivity with an LOD of 100 pM for ssDNA (Fig. 4A), 100 pM for dsDNA (Fig. 4C), and 10 nM for RNA (Fig. 4E). Cas12f dimeric RNP had the sensitivity with an LOD of 1 nM for ssDNA (Fig. 4B), 1 nM for dsDNA (Fig. 4D), and 10 nM for RNA (Fig. 4F). For specificity, SNPs in different locations, including one mismatch (MM) in 5′-end (4-MM in 21 nt), middle (11-MM in 21 nt), and 3′-end (18-MM in 21 nt) of target DNA, were systematically analyzed at the concentration of 1 µM, which will reflect relative differences in specificity between the two systems, rather than a comprehensive positional mapping. Our results showed that both Cas12f functionally monomeric and dimeric RNP are able to distinguish PM with SNP that is in the middle of ssDNA in some degree (ratio_11-MM/PM = 0.6 and 0.77), but not in the 5′-end and 3′-end of ssDNA (Fig. 5A and B). Further experiments showed that, at shorter incubation times, the Cas12f functionally monomeric RNP exhibits a more pronounced difference between the PM and one-mismatch (MM1) targets when using a shorter spacer, compared with the 21-nt ssDNA target (Supplementary Fig. S4). The data suggest enhanced target discrimination under these conditions. As for target dsDNA, the SNP in the middle could be distinguished well from PM by both Cas12f RNPs (ratio_11-MM/PM = 0.14 and 0.15), and SNPs in 5′-end and 3′-end of dsDNA can be distinguished better by Cas12f functionally monomeric RNP (ratio_4-MM/PM = 0.43, ratio_18-MM/PM = 0.39) than by a dimeric RNP (ratio_4-MM/PM = 0.79, ratio_18-MM/PM = 0.87) tracrRNA (Fig. 5C and D). However, as for target RNA, a Cas12f dimeric RNP can distinguish SNP that is in the middle of RNA (ratio_12-MM/PM = 0.18) better than a Cas12f functionally monomeric RNP (ratio_12-MM/PM = 0.33) (Fig. 5E and F). These results demonstrate that Cas12f functionally monomeric RNP not only retains trans-cleavage activity but also exhibits improved sensitivity in detecting ssDNA, dsDNA, and RNA, as well as a slightly improved specificity for target DNA compared to Cas12f dimeric RNP.

Comparison of Cas12f functionally monomeric with dimeric RNPs in gene editing applications

To compare the relative editing performance between functionally monomeric and dimeric Cas12f RNP systems, GFP disruption analysis was evaluated in GFP-positive cell lines (Supplementary Fig. S5A). First of all, Cas9 has been included as a positive control, which showed higher editing efficiency than Cas12f, consistent with previous reports. This confirmed that the reporter system is functional and capable of detecting editing activity (Supplementary Fig. S5B and C). Flow cytometry and confocal microscopy performed 72 h post-transfection revealed similar reductions in GFP-positive cells for both functionally monomeric and dimeric Cas12f RNPs. These observations were consistent across both cell lines, suggesting that the functionally monomeric Cas12f RNP retains comparable editing capability to the dimeric system under the tested conditions (Supplementary Fig. S5B and C).

Discussion

Among Class 2 CRISPR systems, Cas12f is distinguished by its exceptionally small size (400–700 amino acids), offering clear advantages for molecular applications requiring compact and programmable components [14]. However, its native dimeric RNP architecture introduces significant inefficiencies, and only one of the two Cas12f subunits contributes to catalytic activity, resulting in functional redundancy [15, 16]. This not only limits enzymatic efficiency but also increases the molecular size of the RNP complex, complicating its use in applications where minimal design and streamlined delivery are essential.

In this study, we addressed these structural and functional constraints by engineering a monomeric Cas12f RNP through rational guide RNA design. Structural analysis of existing Cas12f complexes (PDB: 7L48 and 7L49) indicated that the 5′-end region (C1–U70) of the tracrRNA is required for dimer formation but not essential for catalytic function. AF3 simulations supported this hypothesis, revealing that truncation of this region prevents binding of a second Cas12f molecule while preserving the core structural framework required for target recognition and cleavage. The resulting monomeric RNP formed a functionally 1:1 Cas12f:gRNA complex with intact catalytic architecture.

Biochemical and cellular assays confirmed that this functionally monomeric Cas12f retains full cis-cleavage activity while exhibiting improved trans-cleavage efficiency (Supplementary Table S3). On the one hand, by eliminating the functionally inactive subunit, the monomeric system ensures that all Cas12f proteins in the reaction contribute directly to target processing, thereby enhancing catalytic performance and maximizing protein utilization. On the other hand, when guiding tracrRNA was truncated, Cas12f RNP was changed from a dimetric to a monomeric form, which may also result in improved accessibility of the active site, or altered RNP population dynamics in solution.

These improvements have important technical implications for CRISPR-based biosensing. The increased trans-cleavage activity enables more sensitive detection of nucleic acids by amplifying signal output, particularly valuable for low copy target analysis. The functionally monomeric RNP also exhibits broad compatibility with a range of conditions, including ambient temperatures and multiple nucleic acid substrates (ssDNA, dsDNA, RNA), facilitating its integration into isothermal amplification workflows or amplification-free diagnostic formats [8, 21–23]. This flexibility, combined with its minimal design, positions functionally monomeric Cas12f as a versatile and efficient sensing element for the development of portable and scalable biosensing platforms.

While the compact size of monomeric Cas12f may eventually support gene editing strategies that benefit from size-restricted delivery systems, such as viral vectors [24], the primary contribution of this study lies in demonstrating a modular strategy to reconfigure CRISPR protein–RNA complexes for enhanced biochemical function. By shifting the Cas12f RNP architecture from dimeric to monomeric through rational truncation of the tracrRNA, we establish a generalizable framework for fine-tuning RNP composition to improve efficiency, activity, and system simplicity.

In conclusion, this work presents a functionally optimized Cas12f monomer RNP with enhanced catalytic performance and minimal complexity. These findings expand the engineering potential of compact CRISPR systems and provide a foundation for developing high-performance molecular tools in biosensing and beyond.

Supplementary Material

gkag430_Supplemental_Files

gkag430_supplemental_files.zip^{(3.1MB, zip)}

Acknowledgements

Author contributions: Lulu Pan (Conceptualization [equal], Data curation [equal], Formal Analysis [equal], Investigation [equal], Methodology [equal], Writing – original draft [equal]), Rui Sang (Data curation [equal], Formal Analysis [equal], Investigation [equal], Writing – review & editing [equal]), Ruier Xue (Data curation [equal], Investigation [equal]), Yongcheng Ma (Writing – review & editing [equal]), Ewa Goldys (Supervision [equal], Writing – review & editing [equal]), and Fei Deng (Conceptualization [equal], and Funding acquisition [equal], Project administration [equal], Supervision [equal], Writing – review & editing [equal])

Contributor Information

Lulu Pan, School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia; ARC Centre of Excellence for Nanoscale Biophotonics, University of New South Wales, Sydney 2052, Australia; Central China Fuwai Hospital of Zhengzhou University, Henan Provincial People’s Hospital, Zhengzhou, Henan 450003, China; Henan Key Laboratory of Individualized Drug Therapy for Cardiovascular Diseases, Zhengzhou, Henan 450003, China.

Rui Sang, School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia; ARC Centre of Excellence for Nanoscale Biophotonics, University of New South Wales, Sydney 2052, Australia.

Ruier Xue, School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia; ARC Centre of Excellence for Nanoscale Biophotonics, University of New South Wales, Sydney 2052, Australia.

Yongcheng Ma, Central China Fuwai Hospital of Zhengzhou University, Henan Provincial People’s Hospital, Zhengzhou, Henan 450003, China; Henan Key Laboratory of Individualized Drug Therapy for Cardiovascular Diseases, Zhengzhou, Henan 450003, China.

Ewa Goldys, School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia; ARC Centre of Excellence for Nanoscale Biophotonics, University of New South Wales, Sydney 2052, Australia.

Fei Deng, School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia; ARC Centre of Excellence for Nanoscale Biophotonics, University of New South Wales, Sydney 2052, Australia.

Supplementary data

Supplementary data is available at NAR online.

Conflict of interest

None declared.

Funding

The authors acknowledge the support of the Henan Province High-level Talent International Exchange Project and Henan Provincial Natural Science Foundation (No. 252300423880) to L.P., UNSW SHARP program to E.G., Early Career Fellowship of Cancer Institute NSW (2024/ECF1573) to F.D., ARC Discovery Project (DP240103024) to E.G. and F.D., and NHMRC Idea Grant (2030464) to E.G. and F.D. Funding to pay the Open Access publication charges for this article was provided by Early Career Fellowship of Cancer Institute NSW (2024/ECF1573).

Data availability

All data are incorporated into the article and its online supplementary material.

References

1. Liu G, Lin Q, Jin S et al. The CRISPR-Cas toolbox and gene editing technologies. Mol Cell. 2022;82:333–47. 10.1016/j.molcel.2021.12.002 [DOI] [PubMed] [Google Scholar]
2. Wang M, Zhang R, Li J. CRISPR/Cas systems redefine nucleic acid detection: principles and methods. Biosens Bioelectron. 2020;165:112430. 10.1016/j.bios.2020.112430 [DOI] [PMC free article] [PubMed] [Google Scholar]
3. Heler R, Samai P, Modell JW et al. Cas9 specifies functional viral targets during CRISPR-Cas adaptation. Nature. 2015;519:199–202. 10.1038/nature14245 [DOI] [PMC free article] [PubMed] [Google Scholar]
4. Chen J, Chen Y, Huang L et al. Trans-nuclease activity of Cas9 activated by DNA or RNA target binding. Nat Biotechnol. 2021; 43:558–568. 10.1038/s41587-41024-02255-41587 [DOI] [PubMed] [Google Scholar]
5. Chen JS, Ma E, Harrington LB et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018;360:436–9. 10.1126/science.aar6245 [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Li S, Cheng Q, Wang J et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 2018;4:20. 10.1038/s41421-018-0028-z [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Li S, Cheng Q, Liu J et al. CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Res. 2018;28:491–3. 10.1038/s41422-018-0022-x [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Deng F, Li Y, Yang B et al. Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing. Nat Commun. 2024;15:1818. 10.1038/s41467-024-46001-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Harrington LB, Burstein D, Chen JS et al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science. 2018;362:839–42. 10.1126/science.aav4294 [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Gootenberg JS, Abudayyeh OO, Lee JW et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356:438–42. 10.1126/science.aam9321 [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Gootenberg JS, Abudayyeh OO, Kellner MJ et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018;360:439–44. 10.1126/science.aaq0179 [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Li H, Dong X, Wang Y et al. Sensitive and Easy-read CRISPR strip for COVID-19 rapid point-of-care testing. CRISPR J. 2021;4:392–9. 10.1089/crispr.2020.0138 [DOI] [PubMed] [Google Scholar]
13. Myhrvold C, Freije CA, Gootenberg JS et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018;360:444–8. 10.1126/science.aas8836 [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Makarova KS, Wolf YI, Iranzo J et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol. 2020;18:67–83. 10.1038/s41579-019-0299-x [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Xiao R, Li Z, Wang S et al. Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR-Cas12f nuclease. Nucleic Acids Res. 2021;49:4120–8. 10.1093/nar/gkab179 [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Takeda SN, Nakagawa R, Okazaki S et al. Structure of the miniature type V-F CRISPR-Cas effector enzyme. Mol Cell. 2021;81:558–70.e3. 10.1016/j.molcel.2020.11.035 [DOI] [PubMed] [Google Scholar]
17. Abramson J, Adler J, Dunger J et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024;630:493–500. 10.1038/s41586-024-07487-w [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Pan L, Ma Y, Sang R et al. Optimization of CRISPR/Cas12f1 guide RNAs using AlphaFold 3 for enhanced nucleic acid detection. Microchem J. 2025;212:113194. 10.1016/j.microc.2025.113194 [DOI] [Google Scholar]
19. Pan L, Wang A, Sang R et al. AlphaFold 3 sheds insights into chemical enhancer-induced structural changes in Cas12a RNPs. Health Nanotechnol. 2025;1:1. 10.1186/s44301-024-00003-z [DOI] [Google Scholar]
20. Mukherjee S, Balius TE, Rizzo RC. Docking validation resources: protein family and ligand flexibility experiments. J Chem Inf Model. 2010;50:1986–2000. 10.1021/ci1001982 [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Vargas AMM, Sinha S, Osborn R et al. New design strategies for ultra-specific CRISPR-Cas13a-based RNA detection with single-nucleotide mismatch sensitivity. Nucleic Acids Res. 2024;52:921–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Tan M, Liao C, Liang L et al. Recent advances in recombinase polymerase amplification: principle, advantages, disadvantages and applications. Front Cell Infect Microbiol. 2022;12:1019071. 10.3389/fcimb.2022.1019071 [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Park J-W. Principles and applications of loop-mediated isothermal amplification to point-of-care tests. Biosensors. 2022;12:857. 10.3390/bios12100857 [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Fage C, Lemire N, Moineau S. Delivery of CRISPR-Cas systems using phage-based vectors. Curr Opin Biotechnol. 2021;68:174–80. 10.1016/j.copbio.2020.11.012 [DOI] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

gkag430_Supplemental_Files

gkag430_supplemental_files.zip^{(3.1MB, zip)}

Data Availability Statement

All data are incorporated into the article and its online supplementary material.

[B1] 1. Liu G, Lin Q, Jin S et al. The CRISPR-Cas toolbox and gene editing technologies. Mol Cell. 2022;82:333–47. 10.1016/j.molcel.2021.12.002 [DOI] [PubMed] [Google Scholar]

[B2] 2. Wang M, Zhang R, Li J. CRISPR/Cas systems redefine nucleic acid detection: principles and methods. Biosens Bioelectron. 2020;165:112430. 10.1016/j.bios.2020.112430 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Heler R, Samai P, Modell JW et al. Cas9 specifies functional viral targets during CRISPR-Cas adaptation. Nature. 2015;519:199–202. 10.1038/nature14245 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Chen J, Chen Y, Huang L et al. Trans-nuclease activity of Cas9 activated by DNA or RNA target binding. Nat Biotechnol. 2021; 43:558–568. 10.1038/s41587-41024-02255-41587 [DOI] [PubMed] [Google Scholar]

[B5] 5. Chen JS, Ma E, Harrington LB et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018;360:436–9. 10.1126/science.aar6245 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Li S, Cheng Q, Wang J et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 2018;4:20. 10.1038/s41421-018-0028-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7. Li S, Cheng Q, Liu J et al. CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Res. 2018;28:491–3. 10.1038/s41422-018-0022-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8. Deng F, Li Y, Yang B et al. Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing. Nat Commun. 2024;15:1818. 10.1038/s41467-024-46001-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9. Harrington LB, Burstein D, Chen JS et al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science. 2018;362:839–42. 10.1126/science.aav4294 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10. Gootenberg JS, Abudayyeh OO, Lee JW et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356:438–42. 10.1126/science.aam9321 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11. Gootenberg JS, Abudayyeh OO, Kellner MJ et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018;360:439–44. 10.1126/science.aaq0179 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Li H, Dong X, Wang Y et al. Sensitive and Easy-read CRISPR strip for COVID-19 rapid point-of-care testing. CRISPR J. 2021;4:392–9. 10.1089/crispr.2020.0138 [DOI] [PubMed] [Google Scholar]

[B13] 13. Myhrvold C, Freije CA, Gootenberg JS et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018;360:444–8. 10.1126/science.aas8836 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14. Makarova KS, Wolf YI, Iranzo J et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol. 2020;18:67–83. 10.1038/s41579-019-0299-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15. Xiao R, Li Z, Wang S et al. Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR-Cas12f nuclease. Nucleic Acids Res. 2021;49:4120–8. 10.1093/nar/gkab179 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. Takeda SN, Nakagawa R, Okazaki S et al. Structure of the miniature type V-F CRISPR-Cas effector enzyme. Mol Cell. 2021;81:558–70.e3. 10.1016/j.molcel.2020.11.035 [DOI] [PubMed] [Google Scholar]

[B17] 17. Abramson J, Adler J, Dunger J et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024;630:493–500. 10.1038/s41586-024-07487-w [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18. Pan L, Ma Y, Sang R et al. Optimization of CRISPR/Cas12f1 guide RNAs using AlphaFold 3 for enhanced nucleic acid detection. Microchem J. 2025;212:113194. 10.1016/j.microc.2025.113194 [DOI] [Google Scholar]

[B19] 19. Pan L, Wang A, Sang R et al. AlphaFold 3 sheds insights into chemical enhancer-induced structural changes in Cas12a RNPs. Health Nanotechnol. 2025;1:1. 10.1186/s44301-024-00003-z [DOI] [Google Scholar]

[B20] 20. Mukherjee S, Balius TE, Rizzo RC. Docking validation resources: protein family and ligand flexibility experiments. J Chem Inf Model. 2010;50:1986–2000. 10.1021/ci1001982 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21. Vargas AMM, Sinha S, Osborn R et al. New design strategies for ultra-specific CRISPR-Cas13a-based RNA detection with single-nucleotide mismatch sensitivity. Nucleic Acids Res. 2024;52:921–39. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Tan M, Liao C, Liang L et al. Recent advances in recombinase polymerase amplification: principle, advantages, disadvantages and applications. Front Cell Infect Microbiol. 2022;12:1019071. 10.3389/fcimb.2022.1019071 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23. Park J-W. Principles and applications of loop-mediated isothermal amplification to point-of-care tests. Biosensors. 2022;12:857. 10.3390/bios12100857 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24. Fage C, Lemire N, Moineau S. Delivery of CRISPR-Cas systems using phage-based vectors. Curr Opin Biotechnol. 2021;68:174–80. 10.1016/j.copbio.2020.11.012 [DOI] [PubMed] [Google Scholar]

PERMALINK

AlphaFold3-guided tracrRNA redesign yields small monomeric Cas12f RNPs

Lulu Pan

Rui Sang

Ruier Xue

Yongcheng Ma

Ewa Goldys

Fei Deng

Roles

Abstract

Graphical Abstract

Graphical Abstract.

Introduction

Materials and methods

Chemicals and biological reagents

Structural simulation of Cas12f RNP

Structural comparison of different complexes

Evaluating trans-cleavage activity of Cas12f RNP with truncated and full-length tracrRNA

Limit of detection test for Cas12f RNP with truncated tracrRNA and full-length tracrRNA

Specificity test for Cas12f RNP with truncated tracrRNA and full-length tracrRNA

In vitro transfection efficiency measurement

Statistical analyses

Results

Structure simulation of Cas12f with truncated and full-length tracrRNA

Figure 1.

Truncation of tracrRNA converts the Cas12f RNP from a dimetric to a functionally monomeric form

Figure 2.

Comparison of Cas12f functionally monomeric RNP with dimeric RNP on biosensing applications

Figure 3.

Figure 4.

Figure 5.

Comparison of Cas12f functionally monomeric with dimeric RNPs in gene editing applications

Discussion

Supplementary Material

Acknowledgements

Contributor Information

Supplementary data

Conflict of interest

Funding

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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