DNA-guided CRISPR–Cas12 for cellular RNA targeting

Carlos Orosco; Boyu Huang; Santosh R Rananaware; August P Bodin; Ivy Browning; Anne Fang; Michael P Baugh; Ian H Lange; Yasmin B Elhabashy; Mahika Balaraju; Jordan G Lewis; Nayan H Shah; Michael P Hanna; Sarah J Flannery; Katelyn S Meister; Vedant Karalkar; Piyush K Jain

doi:10.1038/s41587-026-03129-w

. Author manuscript; available in PMC: 2026 Jul 28.

Published before final editing as: Nat Biotechnol. 2026 May 15:10.1038/s41587-026-03129-w. doi: 10.1038/s41587-026-03129-w

DNA-guided CRISPR–Cas12 for cellular RNA targeting

Carlos Orosco ^1,⁹, Boyu Huang ^1,⁹, Santosh R Rananaware ^1,^2,⁹, August P Bodin ¹, Ivy Browning ², Anne Fang ³, Michael P Baugh ¹, Ian H Lange ¹, Yasmin B Elhabashy ¹, Mahika Balaraju ⁴, Jordan G Lewis ¹, Nayan H Shah ⁵, Michael P Hanna ¹, Sarah J Flannery ¹, Katelyn S Meister ⁶, Vedant Karalkar ⁷, Piyush K Jain ^1,^4,^8,^✉

PMCID: PMC13403136 NIHMSID: NIHMS2179613 PMID: 42141118

Abstract

Here, we present ΨDNA, a DNA-based guide that enables RNA targeting by Cas12 nucleases, overcoming the traditional reliance on RNA-guided systems. We engineer ΨDNA to mimic a CRISPR RNA (crRNA) scaffold in reverse orientation, allowing AsCas12a and Cas12i1 to recognize RNA and trigger strong single-stranded DNA trans-cleavage for sensitive detection of diverse RNA species, including 100% accurate hepatitis C virus RNA detection in clinical samples. ΨDNA also achieves 70–95% multiplex knockdown of endogenous intracellular RNA transcripts through ribosome stalling across multiple human cell lines. Mechanistic studies reveal that activity depends on a stem loop that stabilizes a catalytically competent Cas12–ΨDNA–RNA complex. Lastly, codelivery of crRNA and ΨDNA enables simultaneous DNA editing and RNA knockdown with a single effector and modular fusions of different enzymes to AsCas12a extend ΨDNA to RNase H-mediated RNA degradation and METTL 3-based epitranscriptomic editing. Together, ΨDNA guides constitute an adaptable toolkit that extends Cas12 systems beyond genome editing and diagnostics to enable precise, programmable control of cellular transcriptomes and their epitranscriptomic marks.

Since their discovery, CRISPR–Cas systems have been extensively developed for DNA and RNA genome editing and further engineered for applications such as diagnostics^1–4. Among the most well-known CRISPR–Cas effectors, type II (Cas9) and type V (Cas12) enzymes induce double-stranded breaks in DNA^1,2,5, while type VI (Cas13) targets and degrades RNA^6,7. Additionally, Cas12 and Cas9 exhibit nonspecific collateral cleavage of single-stranded DNA (ssDNA) once the target sequence is bound, while Cas13 has similar activity against RNA. This functionality has been harnessed to develop various nucleic acid detection platforms^3,4,8,9. Overall, these advancements exploit the synergy between Cas effectors and CRISPR RNAs (crRNAs), enabling precise targeting of diverse nucleic acids.

As a part of the CRISPR system, crRNAs have been extensively studied to understand their interactions with Cas proteins^10–12. Transcribed from a CRISPR array, crRNAs are composed of direct repeats and spacer sequences. Once transcribed, the direct repeats are preprocessed to form a 5′ or 3′ handle, which constitutes the scaffold region of the crRNA^5,13,14. In many CRISPR–Cas systems, this scaffold adopts a conserved stem-loop architecture that folds into an RNA pseudoknot structure essential for stable Cas binding and catalytic activation. Thus, the scaffold region stabilizes the crRNA–Cas complex and aids in the recognition and binding of the target, while the adjacent spacer sequence is fully complementary to the target DNA or RNA. Given its unique structure and functionality, numerous efforts have been made to engineer crRNAs to enhance CRISPR–Cas systems. These efforts include incorporating modified bases^15–17, developing chimeric and split crRNAs^18–23 and altering their length^24,25. However, crRNA production is constrained by the complexity and expense of RNA synthesis, along with its shorter shelf life compared to DNA.

Naturally, CRISPR–Cas enzymes work in tandem with the crRNA to stabilize the complex and target a specific sequence^1,2,5. While there have been efforts to modify the target-binding guide region of the crRNA partially or completely with DNA bases for both Cas9 as well as Cas12, previous research has suggested that complete substitution of DNA bases within the guide RNA (sgRNA or crRNA) is typically not well tolerated by both enzyme families^26,27. However, we find the ability of specific type V systems (AsCas12a and Cas12i1) to withstand DNA guides for RNA targeting for cellular and in vitro applications when the guide orientation is reversed. We adapted Cas12 enzymes to adopt synthetic DNA mimics of a guide RNA (termed pseudo-guide DNA or ΨDNA) that assemble with Cas proteins, bind the target RNA and ultimately turn on trans-cleavage activity and have engineered ΨDNAs to detect RNA substrates in a programmable manner.

We first discovered that truncated crRNA with only the target-binding region, retained the trans-cleavage activity of type V CRISPR–Cas systems, specifically Cas12i1 and AsCas12a, against ssDNA targets. We then validated that a complementary DNA (cDNA) sequence matching the target-binding region could activate trans-cleavage in the presence of short RNA targets. To further enhance this activity for RNA targeting, the cDNA was engineered with a 3′ DNA handle to mimic the natural structure of crRNAs, forming what we refer to as ΨDNA. We subsequently leveraged the AsCas12a–ΨDNA complex to induce efficient and specific degradation of various RNA targets in cells by triggering endogenous RNA degradation mechanisms such as ribosome stalling and RNase H1 recruitment. Moreover, this AsCas12a construct was shown to perform dual RNA and DNA targeting, multiplex transcript knockdown and facilitate targeted RNA manipulation when fused to diverse effector proteins, such as RNase H for site-specific cleavage and the RNA methyltransferase METTL3 for programmable RNA modification with N⁶-methyladenosine (m⁶A).

Collectively, the ΨDNA–Cas12 platform provides a versatile toolkit for both high-sensitivity, high-specificity RNA targeting, either alone or in combination with DNA indel formation, enabling precise and programmable post-transcriptional regulation of RNA within living cells.

Results

Engineering of ΨDNAs for RNA detection using Cas12 enzymes

To define how crRNA scaffold length shapes Cas12i1 trans-cleavage, we trimmed the scaffold by 5, 10 or 15 nt and generated a no-scaffold guide RNA (Fig. 1a). Cas12i1 required a near-complete scaffold for potent activity on double-stranded DNA (dsDNA); full-length and minus-five crRNAs were most effective (Fig. 1b). ssDNA targets were still cleaved with the no-scaffold guide RNA, revealing distinct scaffold demands for the two substrates. We next evaluated eight Cas12 orthologs with no-scaffold guide RNA on ssDNA (Fig. 1c). Only Cas12i1 and AsCas12a retained strong activity, implying unique conformational flexibility that could permit DNA guides.

Guided by this observation, we replaced the no-scaffold RNA with 20-nt complementary DNA guides lacking scaffolds and challenged the enzymes with synthetic microRNAs. Only Cas12i1 detected miR-21, miR-122 and miR-155, whereas AsCas12a showed no activity (Extended Data Fig. 1a). To optimize Cas12i1 and enable AsCas12a for a DNA-guided system, we next engineered various structural modifications to the cDNA guide (Fig. 1d). Specifically, we hypothesized that adding DNA mimics of the native crRNA scaffold sequence to the cDNA guide might improve the trans-cleavage activity. We tested four configurations: cDNA alone (no handle), cDNA with a 5′ handle, cDNA with a 3′ handle and cDNA with both 5′ and 3′ handles. Our results demonstrated that the 3′ handle configuration, which we termed ΨDNA, yielded the highest detection activity for both AsCas12a and Cas12i1 enzymes (Fig. 1d). Characterization the substrates involved in ΨDNA-mediated trans-cleavage revealed that AsCas12a retains its specificity for ssDNA (Extended Data Fig. 1b). Using this optimized ΨDNA architecture, we next evaluated additional Cas12 orthologs. Consistent with the earlier results, ΨDNA-mediated trans-cleavage activation was observed only for AsCas12a and Cas12i1 (Fig. 1d and Extended Data Fig. 1f). This structural arrangement likely stabilizes the guide–enzyme complex by positioning the DNA scaffold in a configuration analogous to the native crRNA’s 5′ handle. Interestingly, while Cas12i1 showed generally higher activity with the basic cDNA guide, AsCas12a performed notably better with the optimized ΨDNA structure, suggesting different structural requirements between these enzymes.

We observed excellent specificity as each ΨDNA guide triggered its enzyme only in the presence of the cognate microRNA (Extended Data Fig. 1c–e) and we extended detection to 14 additional microRNAs with similar fidelity (Extended Data Fig. 1g). Optimal guide length ranged from 16 to 28 nt and shorter guides heightened mismatch discrimination (Extended Data Fig. 1i–l). Activity persisted under varied divalent cations, underscoring biochemical robustness (Extended Data Fig. 1h). Electrophoretic mobility shift assays (EMSAs) confirmed stable assembly of AsCas12a with ΨDNA and target RNA (Extended Data Fig. 2a–c). Although AsCas12a exhibits nonspecific RNA binding, we found that nonspecific RNAs compete less effectively with the target RNA in the presence of ΨDNA (Extended Data Fig. 2g). No cis-cleavage or trans-cleavage of RNA was observed, verifying that activation is confined to collateral DNA reporters (Extended Data Fig. 2d,e). In addition, to further assess the mode of interaction among ΨDNA, the target RNA and AsCas12a, DNase I footprinting assays were performed using fluorescently labeled ΨDNA. Protection from DNase I digestion was observed only in the presence of AsCas12a, particularly at the 5′ end (Extended Data Fig. 2f).

Biolayer interferometry measured binding affinities of 3.7 nM for crRNA and 24.5 nM for ΨDNA, indicating slightly weaker yet adequate association (Extended Data Fig. 3a–c). To further characterize the enzyme kinetics of the AsCas12a–ΨDNA–RNA ternary complex, initial velocities were measured and Michaelis–Menten values were determined. We observed that the AsCas12a–ΨDNA system catalyzed trans-ssDNA cleavage at a rate of ~10 turnovers per second, with a catalytic efficiency (k_cat/K_m) of 1.2 × 10⁷ s⁻¹ M⁻¹ (Extended Data Fig. 3d,e). These kinetic parameters are consistent with previously reported values for Cas12a (refs. 4,28).

We then challenged the system with long RNA targets (~1.7 kb) using in vitro transcribed fragments (Fig. 1e). AsCas12a scored on 24/26 ΨDNAs (92.3%), clearly separating controls, while Cas12i1 was active on 9/26 (34.6%). These results indicate that AsCas12a displays increased sequence flexibility. Moreover, validation using mRNA standards with sequences identical to endogenous transcripts demonstrates its potential for endogenous mRNA detection (Extended Data Fig. 4a). Seeking further gains, we screened 77 distinct scaffold sequences as 3′ handles (Fig. 1f,g). Detection efficiency varied widely. AsCas12a performed best with LbCas12a, OpCas12b and its own scaffold; Cas12i1 favored its native sequence and simple AT repeats. For AsCas12a, limits of detection with the top three scaffolds reached 1–10 pM, with the LbCas12a scaffold surpassing the canonical sequence (Extended Data Fig. 4b–e). Additionally, the ΨDNA–Cas12 system demonstrated greater scaffold flexibility than conventional crRNA-guided systems when targeting DNA (Extended Data Fig. 4f,g), which enhanced flexibility enabling guide reengineering in multiple orientations and design dimensions, thereby providing increased tunability and greater potential for further optimization and broader applications. To systematically evaluate the importance of scaffold structure (stem loop) in ΨDNA function, we developed a unique high-throughput sequencing-based mutational screening assay. Toward this, we first constructed a 256-variant ΨDNA library by indexing 4 nt within the stem loop of the LbCas12a scaffold, the best performing candidates for AsCas12a (Fig. 1h). Each ΨDNA variant was then individually incubated with target RNA and AsCas12a in a trans-cleavage reaction without fluorescence quencher (FQ) reporters. Following this initial incubation, all reactions were pooled and further incubated to selectively deplete weakly bound ΨDNAs and a no-Cas control library was used as background. After the next-generation DNA sequencing (NGS) and ranking the enrichment relative to the least retained variant, we found that the original LbCas12a scaffold remained among the top-performing sequences (ranked 2 of 256) (Fig. 1i). Furthermore, depletion levels strongly correlated with the number of mutations within the stem loop, underscoring its essential role in stabilizing the AsCas12a–ΨDNA–RNA ternary complex (Fig. 1j). To validate this high-throughput result, we selected eight ΨDNA variants and confirmed their activities in trans-cleavage assays, which recapitulated the sequencing-based trends (Fig. 1k).

For clinical translation, we built a two-step workflow. Reverse transcription (RT)–PCR followed by T7 transcription produced RNA amplicons that the ΨDNA–Cas12 reaction detected at picomolar levels. Synthetic mimics from hepatitis C virus (HCV), dengue virus and Zika virus were successfully identified (Fig. 1l). Lastly, we evaluated 40 HCV samples, 20 positive and 20 negative, using ΨDNA guides targeting the 5′ untranslated region (5′UTR) and the E2 gene (Fig. 1m,n). The 5′ untranslated ΨDNA guide detected every positive sample across genotypes 1a and 1b. The E2 ΨDNA selectively detected genotype 1a. NGS confirmed viral presence and sequence identity (Extended Data Fig. 4h,i). Overall diagnostic accuracy was 100%, illustrating the promise of ΨDNA-guided Cas12 detection in clinical settings.

Translational repression in HEK293T cells with ΨDNA and AsCas12a

After characterizing the ΨDNA–Cas12 complex in vitro, we proceeded to evaluate its functionality in living cells, aiming for potential in vivo applications. We tested the ability of the DNA-guided AsCas12a complex to bind RNA within a cellular environment by targeting a specific mRNA sequence to induce ribosome stalling, thereby repressing translation, reducing protein synthesis and triggering RNA degradation pathways. To achieve this, we designed a reporter system in which HEK293T cells were cotransfected with two plasmids: one encoding AsCas12a tagged with GFP and the other encoding mCherry. Additionally, we introduced ΨDNA constructs targeting the mCherry mRNA either at the start codon (ΨDNA1 and ΨDNA1+) or downstream (ΨDNA2) to observe the effect on mCherry translation repression (Fig. 2a). The ΨDNA constructs were modified with phosphorothioate ends to prevent exonuclease degradation within cells and ΨDNA1+ included locked nucleic acids (LNAs) flanking the guide region to enhance heteroduplex binding.

Fig. 2 | — a, Schematic representation of cotransfection of AsCas12a–GFP, mCherry and ΨDNA to induce ribosome stalling in HEK293T cells. ΨDNA1 and ΨDNA1+ target the start codon of mCherry mRNA, whereas ΨDNA2 targets the coding region. b, Microscopy images of cells treated with AsCas12a–GFP, mCherry and four different ΨDNA conditions (no guide, ΨDNA1, ΨDNA1+ and ΨDNA2). Individual GFP and mCherry channels are shown, along with the overlay. Fluorescence microscopy was performed in three independent replicates, all showing consistent results. c, Geometric MFI of mCherry for all four ΨDNA conditions with AsCas12a–GFP and GFP only. mCherry MFI was calculated only from GFP-positive cells. Geometric means are used for all calculations. d, MFI of GFP on all GFP⁺ cells. The difference in expression of GFP was nonsignificant throughout all samples compared to the control. A similar GFP MFI demonstrates equal conditions for all samples. Geometric means were used for all calculations. In c,d, statistical analysis for biologically independent replicates (n = 3) was performed using Dunnett’s multiple-comparison test against the control samples. e, mCherry MFI fold change for all ΨDNA constructs with and without AsCas12a. Statistical analysis for biologically independent replicates (n = 3) was performed using Šidák’s multiple-comparison test to compare the effect of AsCas12a on mCherry expression. f, Relative quantification of mCherry mRNA using 2^−ΔΔCt method. *GAPDH* was chosen as the endogenous control. Statistical analysis for biologically independent replicates (n = 3) was performed using Šidák’s multiple-comparison test to compare the effect of AsCas12a on mCherry mRNA level. Each biological replicate had three technical replicates. In e,f, statistical analysis for biologically independent replicates (n = 3) was performed using a one-way ANOVA followed by Dunnett’s multiple comparisons test (two-tailed, α = 0.05), with multiple comparisons corrected using Dunnett’s method. Error bars represent the mean value ± s.e.m. In e,f, statistical analysis for biologically independent replicates (n = 3) was performed using a two-way ANOVA followed by Šidák’s multiple-comparison test (two-tailed, α = 0.05). Each biological replicate had three technical replicates. Error bars represent the mean value ± s.e.m.

To assess the effects of ΨDNA and AsCas12a, we monitored the levels of GFP and mCherry 16 h after transfection using fluorescence microscopy (Fig. 2b). In our initial observations, the control group, which lacked ΨDNA, exhibited higher overall red fluorescence compared to the ΨDNA-treated samples. Among the treated groups, those with ΨDNA targeting the start codon showed a particularly noticeable reduction in red fluorescence intensity.

To further quantify the changes in mCherry production, we subjected the samples to flow cytometry. To ensure that any observed effects were not because of the ΨDNA alone triggering an antisense oligonucleotide (ASO) RNA degradation pathway, we included an additional control group that received GFP only, instead of the AsCas12a–GFP construct. Following flow cytometry, we compared the mean fluorescence intensity (MFI) of mCherry in all GFP-positive cells to determine whether the presence of AsCas12a and ΨDNA led to a reduction in overall red fluorescence intensity. The MFI of cells treated with AsCas12a and ΨDNA was significantly lower, confirming our initial observation that targeting the start codon is more effective (Fig. 2c and Supplementary Fig. 1). While samples treated with ΨDNA alone also showed reduced mCherry levels, the decrease was less pronounced compared to those treated with AsCas12a. To rule out any potential bias because of differences in transfection efficiency of AsCas12a–GFP or GFP alone, we confirmed that the MFI of GFP across all groups remained consistent (Fig. 2d). Comparing the fold change in mCherry MFI across the different ΨDNA constructs relative to the control clearly demonstrated that mCherry expression level was significantly reduced in AsCas12a group. Although ΨDNA alone reduced mCherry fluorescence, particularly with ΨDNA1+, the presence of AsCas12a further enhanced this effect, leading to a significant decrease in protein expression across all constructs (Fig. 2e). Notably, ΨDNA1 exhibited the highest activity when paired with AsCas12a.

To assess RNA expression within the samples, we extracted total RNA and evaluated mRNA expression levels (Fig. 2f). In all samples, the presence of AsCas12a led to a noticeable decrease in mCherry mRNA levels. Given that the ΨDNA complex is incapable of RNA cis-cleavage or trans-cleavage (Extended Data Figs. 1b and 2d,e), we hypothesize that this mRNA degradation is induced by a no-go decay (NGD) mechanism²⁹. NGD is triggered when ribosomes stall and fail to elongate, leading to the degradation of mRNA that cannot produce functional proteins. Additionally, we observed that ΨDNA1+ alone triggered ASO-driven RNA degradation, as indicated by both MFI and mRNA levels, likely because of the strong hybridization induced by the incorporated LNAs. Nonetheless, AsCas12a significantly enhances gene repression, resulting in overall lower protein levels when both the protein and guide are present, confirming the effectiveness of the ΨDNA–Cas12a complex in HEK293T cells.

Efficient endogenous RNA knockdown using ΨDNA-guided CRISPR–Cas12a with low off-target effects

We assessed the ability of the ΨDNA-guided CRISPR–Cas12a system to selectively knock down endogenous RNA transcripts in HEK293T cells. To this end, we measured the relative expression levels of the PPIA, RPL4 and PCSK9 mRNAs following transfection with ΨDNAs specific to these transcripts (ΨPPIA, ΨRPL4 and ΨPCSK9) in combination with either GFP (control) or AsCas12a (Fig. 3a and Extended Data Fig. 5a). In the control cells transfected with GFP, we observed partial knockdown of the target transcripts, potentially because of low-level ASO knockdown activity exerted by ΨDNAs alone. However, cotransfection of AsCas12a with the ΨDNAs led to a significant enhancement in transcript depletion. Overall, the RNA knockdown resulted in a 50–70% reduction in mRNA levels compared to the nontargeting control (NTC). Moreover, similar knockdown effects were reproducibly observed in multiple cancer cell lines, including HeLa, HepG2 and MCF-7 (Extended Data Fig. 7a). These results demonstrate that, while ΨDNA alone can induce partial knockdown, coexpression with AsCas12a substantially increases the efficiency and specificity of RNA transcript depletion, confirming the dependence of ΨDNA-mediated knockdown on the presence of AsCas12a. Importantly, we observed comparable knockdown efficiencies when ΨDNA was coexpressed with either wild-type AsCas12a or a catalytically inactive mutant (dAsCas12a), further supporting that ΨDNA-mediated RNA knockdown does not arise from direct enzymatic RNA cleavage by AsCas12a (Extended Data Fig. 5g).

Fig. 3 | — a, Schematic representation and relative mRNA expression following cotransfection of AsCas12a–GFP and GFP with ΨDNA (targeting and nontargeting) to induce RNA knockdown. Relative quantification of *PPIA*, *RPL4* and *PCSK9* mRNA in HEK293T cells expressing either GFP (gray, control) or AsCas12a (blue) with ΨNT or gene-specific ΨDNAs (Ψ*PPIA*, Ψ*RPL4* or Ψ*PCSK9*). A significant reduction in target gene expression was observed with ΨDNA-mediated knockdown when codelivered with AsCas12a, demonstrating the specificity and efficiency of the RNA knockdown using the ΨDNA-guided CRISPR–Cas12a system. Statistical analysis was performed using a two-way ANOVA with Šidák’s multiple-comparison test (two-tailed, α = 0.05). b, RIP results of PPIA RNA enrichment in cells expressing HA-tagged AsCas12a compared to HA–GFP control. The RNA fold change quantification indicates RNA targeting by the AsCas12a–ΨDNA complex. Statistical analysis was performed using an unpaired two-tailed t-test. c,d, Volcano plots from mRNA-seq data comparing the off-target effects of AsCas12a–ΨDNA, ΨDNA and RfxCas13d for *PPIA* gene. Each set is compared to a dCas13d crRNA NTC to evaluate gene expression differences among different samples. Plots and statistical analyses were performed in DESeq2 with 95% confidence interval (CI) with n = 2 biologically independent replicates with 30 million reads each. All colored (blue) points represent genes that show statistical significance in downregulation (left) or upregulation (right) with the gene of interest (*PPIA*) are labeled. The exact quantification and comparison are displayed to evaluate the difference in off-target effects among each different condition. DESeq2 (negative binomial model) was applied with the Wald test and Benjamini–Hochberg procedure adjustment; the volcano plot shows the log₂ fold change versus −log₁₀ false discovery rate (FDR). e,f, mRNA-Seq volcano plots and off-target quantification for *RPL4* gene comparing AsCas12a–ΨDNA, ΨDNA and RfxCas13d. Plots and statistical analyses were performed in DESeq2 with 95% CI with n = 2 biologically independent replicates with 30 million reads each. DESeq2 (negative binomial model) was applied with the Wald test and Benjamini–Hochberg procedure adjustment; the volcano plot shows the log₂ fold change versus −log₁₀ FDR. g, Schematic representation and relative mRNA expression in HEK293T cells transduced with AsCas12a–GFP. h, RNA knockdown evaluated with relative quantification of *PPIA*, *RPL4*, *PCSK9*, *NRAS* and *SMARCA4* mRNA with ΨNT (gray) or gene-specific ΨDNAs (blue). i, Volcano plot from Ribo-seq data, comparing AsCas12a with ΨDNA–*PPIA* and the ΨDNA–NT group. Ribosomal occupancies were calculated by RiboDiff, while explicitly accounting for RNA-seq data. TE, translation efficiency. Colored dots represent the statistical significance with 90% CI with n = 2 biologically independent replicates and 100 million reads each. RiboDiff (negative binomial model) was applied with the likelihood ratio test and Benjamini–Hochberg procedure adjustment; the volcano plot shows the log₂ translation efficiency change versus −log₁₀ FDR. j,k, Read coverage of CLIP-seq data. Read counts are normalized by total read number. Green arrows represent the ΨDNA position. BigWig files were visualized using the Integrated Genomics Viewer. AsCas12a–ΨPPIA exhibits higher relative peaks at the *PPIA* and *PPIAP22* transcripts compared to ΨNT: *PPIA* locus (j) and *PPIAP22* locus (k). In a,b,h, bars represent the mean value ± s.e.m. of n = 3 biological replicates.

To validate that AsCas12a is specifically recruiting and binding target RNA transcripts in the presence of ΨDNAs, we conducted RNA immunoprecipitation (RIP) using an HA-tagged version of AsCas12a. Cells delivered with either HA–GFP (control) or HA–AsCas12a and ΨPPIA were subjected to RIP, followed by qPCR analysis of the immunoprecipitated RNA to quantify the binding (Fig. 3b). We observed that PPIA transcripts were significantly enriched in samples expressing HA–AsCas12a compared to those expressing the control HA–GFP, demonstrating that AsCas12a binds directly to its targeted PPIA transcript when guided by ΨDNA. These results confirm that ΨDNA is facilitating direct interaction between AsCas12a and its endogenous RNA targets.

To assess the flexibility of ΨDNA, we designed multiple target sites in PPIA, RPL4 and PCSK9 on the basis of SHAPE scores from the RASP version 2.0 database and found no significant correlation between RNA knockdown efficiency and local RNA secondary structure^30–32. Using CLIP-seq data from the POSTAR3 database, we further designed six target sites overlapping RNA-binding protein (RBP) binding regions in PPIA and RPL4 transcripts³³. RBPs influenced ΨDNA-mediated RNA knockdown at PPIA sites but had little effect at RPL4 sites, indicating that RBP effects on ΨDNA targeting are target dependent (Extended Data Fig. 5b–f).

To further evaluate the cellular feasibility of the AsCas12a–ΨDNA complex, we systematically assessed the off-target effects of this therapeutic strategy. We further hypothesized that, because of the absence of RNA trans-cleavage activity in AsCas12a, modulation of the target transcript by this system is likely to impose fewer perturbations on other cellular pathways. We performed mRNA-seq to quantify the off-target effects of the AsCas12a–ΨDNA complex and ΨDNA alone and compared them to RfxCas13d (Fig. 3c–f and Extended Data Fig. 6a–d) for two targets transcribed by PPIA and RPL4 genes. We observed that, compared to ΨDNA alone, the AsCas12a–ΨDNA complex not only achieved greater RNA knockdown efficiency but also exhibited 2–7-fold lower off-target effects. This suggests that AsCas12a has a notable role in guiding target binding and enhancing specificity in the presence of the DNA guide. Furthermore, while AsCas12a–ΨDNA exhibited lower knockdown efficiency compared to RfxCas13d (Extended Data Fig. 5h), AsCas12a showed substantially lower off-target effects than RfxC-as13d for both targets: 17.7-fold lower for PPIA and 6.3-fold lower for RPL4. These results clearly demonstrate that the absence of RNA trans-cleavage significantly reduces off-target effects and the AsCas12a–ΨDNA complex is evidently less toxic to the transcriptome compared to the widely used RfxCas13d.

To increase RNA knockdown efficiency, we determined that having AsCas12a already present in the cell at the time of ΨDNA transfection is more effective, as the ΨDNAs can immediately complex with the protein and avoid degradation in the cytoplasm. To enable this, we generated a lentiviral cell line that expresses AsCas12a and GFP on the same transcript. In this cell line, we tested the transfection of DNA guides targeting five different transcripts: PPIA, RPL4, PCSK9, NRAS and SMARCA4 (Fig. 3g,h). We observed substantial improvements in knockdown efficiency, with PPIA and RPL4 expression reduced by 95% and 80%, respectively. Similarly, PCSK9 and NRAS showed robust knockdown, with a ~90% reduction in gene expression. By contrast, SMARCA4 exhibited a more moderate knockdown at around 60%. Overall, AsCas12a transduction significantly enhanced the efficiency of the ΨDNA complex.

To determine whether ΨDNA-mediated RNA knockdown is associated with translational repression, we performed Ribo-Seq paired with RNA-Seq on cells cotransfected with AsCas12a and ΨPPIA or a nontargeting ΨDNA control (ΨNT) (Fig. 3i and Extended Data Fig. 6e,f). The analysis revealed the target PPIA had increased ribosomal occupancy and a total of 17 transcripts exhibited significant changes in translational efficiency, including the intended PPIA and its homologous transcripts PPIAL4C and PPIAL4G. These results indicate that ΨDNA-mediated RNA knockdown is accompanied by highly selective translational effects and is associated with minimal off-target perturbation. Taken together, these results provide evidence that ΨDNA–AsCas12a induces ribosomal stalling consistent with an NGD-like mechanism, while maintaining specificity at the translational level.

Additionally, we performed CLIP-seq to obtain a more in-depth and granular view of enzyme binding in a cellular context. Following sequencing and data analysis, we plotted normalized CLIP-seq peaks to compare binding profiles between ΨDNAs (nontargeting and targeting) in the presence of AsCas12a (Fig. 3j,k). As shown, binding of the enzyme at the PPIA locus targeted by ΨDNA is highly enriched relative to the NTC, demonstrating strong target specificity in cells. We also observed enrichment at the PPIAP22 pseudogene, which shares an identical target sequence to PPIA, further indicating that binding is driven by sequence complementarity. Additionally, we observed similar results with ΨMix transfection (targeting PPIA, RPL4, SMARCA4 and PCSK9) (Extended Data Fig. 6g), showing multiple peaks for the different targeted transcripts.

Potential application of AsCas12a and ΨDNA system

We next established a ΨDNA-based platform for simultaneous DNA editing and RNA targeting using a single CRISPR effector, extending the application of ΨDNA beyond the conventional functional scope of Cas12-based or Cas13-based systems. By codelivering ΨDNA and crRNA, this strategy enables concurrent RNA knockdown and DNA indel formation (Fig. 4a). To achieve this, we delivered AsCas12a lacking a nuclear localization signal (NLS), together with either ΨPPIA or ΨRPL4 and a crRNA targeting the CCR5 locus. After 18 h, RNA levels were reduced for ΨPPIA and ΨRPL4 relative to the ΨNT control (Fig. 4b). Although we observed statistical significance between the ΨRPL4–GFP and ΨRPL4–AsCas12a conditions, no significant difference was observed for the ΨPPIA datasets. To determine whether AsCas12a influenced RNA knockdown specificity, we performed mRNA-seq on these samples to evaluate off-target effects and assess whether the presence of the protein improves RNA-targeting specificity (Fig. 4b,c). The data show that samples containing AsCas12a exhibited a total of 14 off-target transcripts, compared to 605 off-targets in the ΨPPIA-only condition, corresponding to an approximately 43-fold reduction. In parallel, amplicon sequencing confirmed efficient genome editing on the CCR5 locus at 18 and 50 h after transfection, with indel frequencies of approximately 10–15% (Fig. 4d and Supplementary Fig. 3). Together, these data demonstrate that the AsCas12a–ΨDNA system enables dual DNA–RNA targeting using a single effector, providing a versatile and previously unreported approach for coordinated regulation of genomic DNA and endogenous RNA.

Fig. 4 | — The ΨDNA–AsCas12a platform enables dual DNA–RNA targeting, multiplexed translational repression, targeted RNA degradation and epitranscriptomic editing. a, Schematic representation of the cotransfection of ΨDNA, crRNA and protein expression plasmids encoding AsCas12a–GFP or GFP. b, Relative quantification of *PPIA* and *RPL4* mRNA in HEK293T cells following cotransfection of both ΨDNA and crRNA. GFP (gray, control) or AsCas12a (blue) and ΨNT or gene-specific ΨDNAs (Ψ*PPIA* or Ψ*RPL4*) were detected. c, Volcano plots from mRNA-seq data comparing the off-target effects of AsCas12a–ΨDNA + crRNA and ΨDNA + crRNA for *PPIA* gene. Plots and statistical analyses were performed in DESeq2 with 95% CI with n = 2 biologically independent replicates. DESeq2 (negative binomial model) was applied with the Wald test and Benjamini–Hochberg procedure adjustment; the volcano plot shows the log₂ fold change versus −log₁₀ FDR. d, Genome-editing efficiency on *CCR5* locus from NGS data. The no-treatment control and transfection groups (ΨDNA only, crRNA only and both ΨDNA and crRNA) are shown on the plot with 18 h (gray) or 50 h (green) of editing. e, Schematic representation and relative mRNA expression in HEK293T cells transduced with AsCas12a–GFP. RNA knockdown was evaluated by relative quantification of *PPIA*, *RPL4*, *PCSK9*, *NRAS* and *SMARCA4* mRNA with ΨNT (gray) or gene-specific ΨDNAs (blue). f, Schematic representation and multiplex knockdown of four endogenous transcripts (*PPIA*, *RPL4*, *NRAS* and *PCSK9*) using ΨNT or various ΨDNA combinations as described (Ψ*PPIA*, ΨMix II, ΨMix III and ΨMix IV), showing differential and specific knockdown efficiency across gene targets in HEK293T cells transduced with AsCas12a. Results highlight the potential for multiplex RNA targeting using the ΨDNA-guided CRISPR–Cas12a platform. g, Microscope images after PLA. Green fluorescence represents GFP coexpressed with Cas proteins. The Texas red signal represents RNase H1 colocalized with Cas protein inside HEK293T cells. h, Schematic representation showing RNase H1 fused to the N terminus of AsCas12a; the fusion protein can be guided by ΨDNA and induce RNA degradation. Fluorescence microscopy was performed in three independent replicates, all showing consistent results. i, Relative mRNA expression for cotransfection of C–GFP, AsCas12a–C–GFP, AsCas12a–GFP or GFP and ΨDNA (targeting and nontargeting) to induce RNA knockdown. With relative quantification of *RPL4* mRNA in HEK293T cells, the AsCas12a–C group shows higher RNA knockdown compared to WT AsCas12a–GFP and C–GFP. j, Schematic representation showing the RNA methyltransferase METTL3 fused on the C terminus of AsCas12a; the fusion protein can be guided by ΨDNA and induce target RNA methylation. k, Relative RNA methylation is detected by MeRIP–qPCR for METTL3–AsCas12a or AsCas12a in HEK293T cells. The METTL3–AsCas12a group shows a significantly increased m⁶A level. In d,f, bars represent the mean value ± s.e.m. of n = 3 biological replicates. In b,i,k, statistical analyses for biologically independent replicates (n = 3) were performed using a two-way ANOVA (two-tailed, α = 0.05) followed by Šidák’s multiple-comparison test. Error bars represent the mean ± s.e.m.

To evaluate the capability of ΨDNA-guided CRISPR–Cas12a for multiplex RNA transcript knockdown, we next codelivered combinations of ΨDNAs targeting multiple endogenous transcripts, including PPIA, RPL4, NRAS and PCSK9. We transfected different ΨDNA combinations in the AsCas12a-transduced cell line, including ΨPPIA (targeting PPIA only), ΨMix II (targeting PPIA and RPL4), ΨMix III (targeting PPIA, RPL4 and NRAS) and ΨMix IV (targeting PPIA, RPL4, NRAS and PCSK9) (Fig. 4e,f). We observed that individual ΨDNAs and multiplexed combinations (ΨMix II, ΨMix III and ΨMix IV) achieved efficient transcript depletion across all target RNAs. Specifically, ΨMix IV resulted in robust knockdown of +70% for PPIA, RPL4, NRAS and PCSK9 simultaneously, highlighting the potential of this system for targeting multiple transcripts in parallel. Multiplexed RNA knockdown was also achieved with AsCas12a transfection, resulting in approximately 50% reduction in expression across four simultaneous target genes (Extended Data Fig. 7e).

Collectively, these findings demonstrate that the ΨDNA-guided CRISPR–Cas12a system enables precise and efficient depletion of endogenous RNA transcripts, both individually and in a multiplexed manner. The system offers considerable versatility for RNA-targeting applications and holds strong potential for therapeutic interventions aimed at reducing disease-relevant RNA transcripts. Its low off-target effects and high knockdown efficiency make AsCas12a a particularly promising platform for therapeutic use, especially in contexts where preserving transcriptome integrity is essential.

We further investigated whether RNA knockdown mediated by the AsCas12a–ΨDNA system involves additional cooperative pathways beyond a single mechanism. To test whether RNase H1 is spatially associated with the ΨDNA–AsCas12a complex during RNA targeting, we performed a proximity ligation assay (PLA). GFP–AsCas12a–ΨPPIA and GFP–dRfxCas13d–ΨPPIA constructs were analyzed using antibodies to the HA tag and endogenous RNase H1. PLA signals, detected as red fluorescence, indicate close spatial proximity between the protein and RNase H1. Notably, AsCas12a-expressing cells exhibited a strong PLA signal, whereas dRfxCas13d showed minimal signal (Fig. 4g). This finding suggests that AsCas12a–ΨDNA-mediated RNA degradation may also involve RNase H1, beyond the NGD mechanism previously characterized.

Accordingly, we explored whether directly fusing RNase H1 to AsCas12a could further enhance RNA knockdown efficiency (Fig. 4h). To compare the effects of RNase H1-fused AsCas12a and AsCas12a alone, we cotransfected a pCMV expressing mCherry plasmid and ΨDNA2 previously characterized (Fig. 2b). Fluorescence microscopy showed that, similar to AsCas12a, RNase H1-fused AsCas12a significantly reduced mCherry signal when targeted by ΨDNA2 (Extended Data Fig. 7c). To assess changes at the RNA level, we isolated the total RNA from the experimental groups and performed RT–qPCR. The results indicated that ΨDNA2 induced a significant reduction in mCherry RNA levels when RNase H1-fused AsCas12a was expressed, compared to the NTC (Extended Data Fig. 7d). RNA knockdown was still observed with wild-type AsCas12a and GFP, but to a lesser extent. To minimize potential effects arising from RNase H1 overexpression, using ΨDNA targeting the endogenous RPL4 transcript, we observed that cotransfection of the RNase H1 C-terminal domain (RNase H1 C) did not induce measurable RPL4 knockdown, in contrast to full-length RNase H1 (Extended Data Fig. 7b). In light of the observations above, we fused RNase H1 C to AsCas12a to enhance RNA knockdown of an abundant endogenous transcript such as RPL4 (Fig. 4i). The fused construct lowered the RNA levels ~15% more than normal AsCas12a–ΨDNA, showing significant statistical improvement. Additionally, the data demonstrate that the RNase H–AsCas12a fusion construct effectively reduced RPL4 RNA levels in a manner dependent on guidance by the AsCas12a enzyme, as only the C terminus was added.

In addition, m⁶A is the most abundant internal RNA modification, known to regulate RNA secondary structure, alternative splicing and stability³⁴. To evaluate site-specific epitranscriptomic editing, we fused the m⁶A methyltransferase METTL3 to AsCas12a and targeted it to RNA using ΨDNA (Fig. 4j). We cotransfected METTL3-fused AsCas12a or AsCas12a alone with ΨDNAs targeting two sites: A690 in GAPDH and A3488/A3504 in FOXM1.

Methylated RIP (MeRIP)–qPCR analysis revealed that, upon targeting by ΨGAPDH or ΨFOXM1, METTL3-fused AsCas12a induced a significant increase in m⁶A modification at the target sites compared to the NT group, whereas wild-type AsCas12a alone had no detectable effect (Fig. 4k). These results demonstrate that AsCas12a, when fused to functional effector proteins such as METTL3, can be guided by ΨDNA to achieve site-specific manipulation of RNA.

Discussion

Consistent with the conformational rearrangements observed when Cas proteins bind crRNA followed by target DNA recognition, which move the RuvC domain to its active position and thereby enable trans-cleavage activity^1,8,14,35, we propose that a similar structural transition occurs upon formation of the AsCas12a–ΨDNA–RNA ternary complex. The formation of this complex may likewise induce conformational changes and activate collateral activity. This mechanism parallels how crRNA–dsDNA engagement triggers nonspecific ssDNA cleavage. Moreover, the trans-cleavage activity of both enzymes, AsCas12a and Cas12i1, is greatly increased when the DNA guide has a 3′ handle. This evidence suggests ΨDNA further stabilizes the Cas protein complex, which is translated into a higher fluorescence signal. Additionally, we observed no cis-cleavage or trans-cleavage of RNA, as Cas12 enzymes do not possess a HEPN domain capable of RNA degradation like Cas13 (ref. 36). Instead, in most Cas12 enzymes, the RuvC domain is primarily associated with DNA cleavage, except for Cas12g and Cas12a2.

Although ΨDNAs cannot be genetically encoded or expressed from plasmids, current methods for RNA synthesis include solid-phase synthesis, in vitro transcription (IVT) and other enzymatic processes. Solid-phase synthesis of RNA is more expensive and time-consuming than that of DNA, requiring costly reagents, specialized chemistry and extensive steps³⁷. IVT involves an initial DNA synthesis step, followed by enzymatic processes to produce RNA, adding to the overall cost and complexity. Additionally, RNA has a shelf life that is several orders of magnitude shorter than that of DNA, necessitating low temperatures or specialized storage conditions to maintain stability. CRISPR–Cas nucleic acid detection platforms, such as SHERLOCK and DETECTR^3,4, predominantly rely on crRNA, making these technologies more expensive and less accessible. This efficiency in time and cost is particularly advantageous in high-throughput applications to allow faster iteration of guides and optimize experimental conditions. Thus, the use of ΨDNA represents a practical and economical alternative for large-scale studies involving RNA detection and targeting. A recent publication further validated our initial findings^38,39.

Most importantly, while CRISPR–Cas12 systems have been extensively engineered for DNA genome editing in cells, we successfully demonstrate a unique RNA-targeting construct that greatly expands the system’s applicability to RNA gene regulation. Our study shows that AsCas12a can complex with ΨDNA to target mRNA within cells, leading to reduced mRNA levels and decreased protein production. As this DNA-guided system lacks RNA cis-cleavage or trans-cleavage activity, we found that the ΨDNA–AsCas12a complex exhibits lower off-target effects than RfxCas13d, a widely used RNA-targeting enzyme. The reduced off-target toxicity of DNA-guided AsCas12a presents a strategy for transient gene silencing and holds promise for RNA editing within cells, similar to approaches using catalytically inactive Cas13 variants^40–42.

Furthermore, we demonstrate that the ΨDNA–AsCas12a platform enables simultaneous DNA and RNA targeting using a single CRISPR effector. The dual RNA–DNA targeting capability enables multilayer gene regulation by combining immediate transcript suppression with permanent genomic disruption. This strategy may accelerate functional genomics studies by distinguishing early transcript-level effects from long-term genomic consequences while reducing compensatory cellular responses. Additionally, integrating both functionalities simplifies delivery compared to approaches requiring separate RNA interference and genome-editing components, expanding opportunities in multiplexed gene regulation, synthetic biology and therapeutic development.

In therapeutic contexts, this approach could simultaneously silence pathogenic RNAs while editing the underlying DNA source. For example, while CCR5 knockout is a well-established strategy to prevent HIV entry, viral tropism switching to CXCR4 remains a limitation. Because CXCR4 is essential for hematopoietic stem cell homing, permanent disruption is lethal. In this scenario, our system could leverage crRNA-mediated CCR5 disruption while ΨDNA transiently suppresses CXCR4 mRNA, potentially limiting viral escape without eliminating an essential gene. Although further engineering and delivery optimization will be required to evaluate such applications in disease models, these results highlight the potential of coordinated RNA and DNA targeting by a single nuclease.

Additionally, we showcased multiplexed gene silencing across four different targets, highlighting the platform’s potential for high-throughput applications akin to previous Cas13-based screens⁴³. In our multiplex knockdown system, the ΨMix IV group, designed to simultaneously target four distinct transcripts, achieved robust and efficient knockdown, with efficiencies ranging from 70.7% to 81.9%. These findings underscore the potential of the ΨDNA–AsCas12a platform as a scalable and cost-effective approach for multiplexed CRISPR screening. Additionally, the ability to coordinately modulate multiple gene expressions offers a promising strategy for advancing RNA-targeted therapeutics.

As for the mechanism of ΨDNA–AsCas12a-mediated RNA knockdown, our observations indicate that targeting by the ΨDNA–AsCas12a complex is associated with changes in translational output of the target RNA and that the complex exhibits spatial proximity to endogenous RNase H1. These findings are consistent with the possibility that multiple regulatory processes contribute to RNA reduction. Lastly, we demonstrated that AsCas12a fusion proteins, specifically with RNase H and METTL3, can mediate precise and programmable regulation of target RNAs when guided by ΨDNA. The inherent modularity of AsCas12a enables its fusion with a variety of functional protein domains, allowing tailored applications in RNA biology. These include but are not limited to locus-specific RNA labeling, targeted base editing and other epitranscriptomic modifications in cells, thereby broadening the scope of RNA-targeted interventions at the post-transcriptional level.

Methods

Plasmid construction

Plasmids encoding Lb, As, Er and other Cas12a variants, as well as BrCas12b, were constructed following previously described protocols^{22,24,44–47}. Escherichia coli codon-optimized Cas12i1 and Cas12i2 plasmids were obtained from Addgene (Arbor Biotechnologies, 120882 and 120883). For mammalian expression, the NLS was removed from AsCas12a-P2A-GFP (Addgene, 160140) by KLD mutagenesis. The mCherry construct was generated by inserting the coding sequence into a pCMV vector lacking the CMV enhancer. RfxCas13d, PspCas13b (derived from Addgene, 155367) and DisCas7-11 (Addgene, 172507) were cloned into a common backbone.

For lentiviral expression, AsCas12a-P2A-GFP was subcloned into the lentiCas9-Blast vector (Addgene, 52962) using NEBuilder HiFi DNA assembly (New England Biolabs (NEB), E2621L). RNase H1 was amplified from ppyCAG-RNase H1-WT (Addgene, 111906) and fused to the N terminus of AsCas12a, whereas METTL3 together with NLS was amplified from pCMV-dCas13-M3nls (Addgene, 155366) and fused to the C terminus.

Protein expression and purification

Rosetta (DE3) cells harboring expression plasmids were grown on agar plates at 37 °C overnight. Single colonies were inoculated into 10 ml of Luria–Bertani medium and cultured for ~12 h, followed by expansion into 1.5 L of Terrific Broth medium. Cells were grown to an optical density at 600 nm of 0.6–0.8, cooled for 45–60 min, induced with 0.5 mM IPTG and expressed at 16 °C for 14–18 h.

Cells were harvested by centrifugation and resuspended in lysis buffer (500 mM NaCl, 50 mM Tris-HCl pH 7.5, 20 mM imidazole, 0.5 mM TCEP, 1 mM PMSF, lysozyme and DNase I). Lysates were clarified by sonication and high-speed centrifugation, filtered (0.22 μm) and loaded onto a HisTrap FF column (Co²⁺-charged) using a fast protein liquid chromatography system. Proteins were eluted with imidazole-containing buffer (250 mM imidazole).

Except for Cas12i1 and Cas12i2, fractions were pooled, treated with TEV protease and dialyzed overnight at 4 °C in buffer (500 mM NaCl, 50 mM HEPES pH 7, 5 mM MgCl₂ and 2 mM DTT). Samples were concentrated and further purified by heparin affinity chromatography followed by size-exclusion chromatography (Superdex 200). Peak fractions were pooled, concentrated, flash-frozen in liquid nitrogen and stored at −80 °C.

Oligonucleotide preparation

All ssDNA and RNA oligos including guide RNA, guide DNA, target activators, primers and fluorescent reporters were obtained from Integrated DNA Technologies (IDT) and diluted in 1× TE buffer (10 mM Tris and 0.1 mM EDTA, pH 7.5). For generating long RNA target mimics of HIV, Zika virus, dengue virus and HCV RNA, dsDNA gene fragments containing a T7 promoter region were ordered from Twist Biosciences and in vitro transcribed using HiScribe T7 high-yield RNA synthesis kit (NEB, E2040S) to generate the long RNA fragments.

CRISPR–Cas-based fluorescence detection assay

Fluorescence-based detection assays were performed in black 384-well plates. crRNA–Cas12 complexes were assembled in NEB buffer 2.1, incubated at room temperature for 10 min and then combined with FQ reporter (250–500 nM) and target activator in a total volume of 40 μl. Reactions were incubated at 37 °C and fluorescence was measured using a microplate reader (excitation: 483/20 nm, emission: 530/20 nm) at 2.5-min intervals. Unless otherwise indicated, final concentrations were 50 nM Cas enzyme, 100 nM crRNA or ΨDNA and 25 nM target.

For Michaelis–Menten analysis, AsCas12a–ΨDNA–RNA complexes were assembled as above and diluted to a final effective complex concentration of 1.25 nM (50 nM AsCas12a, 1.25 nM ΨDNA and 50 nM target RNA). Reactions were performed in NEB buffer 2.1 with increasing concentrations of FQ reporter (0.01–2 μM). Fluorescence was recorded at 37 °C using a real-time PCR system at 13-s intervals. A standard curve was generated using FAM reporter without quencher. Initial velocities (V₀) were determined by linear regression and kinetic parameters were calculated using Prism10.

EMSA

EMSA gel was performed by mixing 100 nM of Cas12 protein (AsCas12a or Cas12i1), 100 nM guide (crRNA or ΨDNA) and 100 nM target (ssDNA or ssRNA) in NEB 2.1 buffer and incubating the reaction at 4 °C for 30 min. Then, 1 μl of 5× TBE Hi-Density sample buffer (Invitrogen, LC6678) was added to 9 μl of sample. The samples were loaded in a native PAGE DNA retardation gel (Invitrogen, EC6365BOX) and run at 200 V for 30 min. Later, PAGE gel was stained with SYBR gold (Invitrogen, S11494) and imaged on Amersham Typhoon.

Biolayer interferometry

Biolayer interferometry was performed on a GatorBio system using streptavidin biosensors (Flex SA Kit) and 3′-biotinylated DNA or RNA guides. Assays were conducted in NEB buffer 2.1 supplemented with 0.05% Triton X-100. Guides were immobilized at 50 nM onto streptavidin probes before measurement.

Association was measured by transferring loaded sensors into wells containing AsCas12a at concentrations of 1, 2.5, 5, 10, 25, 50 and 100 nM for 10 min. Dissociation was monitored by transferring sensors into buffer-only wells for an additional 10 min.

Data were analyzed using GatorBio software. Binding curves were fitted to a 1:1 binding model to determine association (k_on) and dissociation (k_off) rate constants. Equilibrium dissociation constants (K_d) were derived from steady-state analysis of concentration-dependent responses. Sensors without immobilized guides were used as negative controls.

Systematic ΨDNA evaluation assay

All ΨDNAs were synthesized by IDT. First, 50 nM AsCas12a,100 nM target RNA and 200 nM ΨDNA were incubated at 37 °C for 30 min; then, all ΨDNA groups were pooled together into a 15-ml tube and incubated at 37 °C for another 30 min. After incubation, pooled ΨDNAs were diluted 100-fold for amplicon library preparation. Q5 DNA polymerase was used for PCR. A library lacking AsCas12a was used as a control.

The amplicon libraries were sequenced by Illumina NextSeq 500 with a NextSeq 500/550 mid output kit v2.5 (150 cycles) (Illumina, 20024904). All computational work was performed on the University of Florida (UF) high-performance computing (HPC) cluster HiperGator.

IVT

IVT of long HIV and HCV RNA fragments was performed using a HiScribe T7 high-yield RNA synthesis kit (NEB, E2040S) following the manufacturer’s protocol and purified using the Monarch RNA cleanup kit (NEB, T2030L).

Endogenous mRNA detection

Total RNA from 1 × 10⁶ HEK293T cells (American Type Culture Collection (ATCC), CRL-3216) was extracted using the Monarch total RNA miniprep kit (NEB, T2010S). Then, cDNA was synthesized using the PhotoScript II first-strand cDNA synthesis kit (NEB, E6560) with Oligo-dT primers. Lastly, 17 endogenous genes were amplified using KOD One PCR master mix (Toyobo, KMM-201) and in vitro transcribed as described above. For detection, 2 μl of product was added to a CRISPR–Cas-based fluorescence detection assay.

Participant sample collection

The collection and processing of participant samples were approved by the UF Institutional Review Board (IRB202200294). For clinical validation, a total of 20 human serum samples were obtained from participants with HCV collected under the HCV-TARGET program, which also supplied the UI per ml of each sample reported in Fig. 4. Healthy serum samples were obtained from Boca Biolistics.

Participant sample extraction

Viral RNA was extracted from serum samples using the Quick-DNA/RNA viral MagBead kit (Zymo, R2140) according to the manufacturer’s instructions with minor modifications. Briefly, 200 μl of serum was treated with 10 μl of proteinase K (20 mg ml⁻¹) for 15 min at room temperature, followed by the addition of DNA/RNA Shield (1:1, v/v). Samples were mixed with 800 μl of viral DNA/RNA buffer and 20 μl of MagBinding beads, vortexed for 10 min and separated using a magnetic rack.

Beads were washed sequentially with 250 μl of wash 1, 250 μl of wash 2 and two washes with 250 μl of 100% ethanol. After air-drying for 10 min, RNA was eluted in 30–60 μl of nuclease-free water and used for downstream analysis.

Participant sample validation

After extraction, samples were amplified by adding 1 μl of extracted viral RNA to a 50-μl SuperScript IV one-step RT–PCR reaction (Invitrogen, 12594025) and thermocycling was performed according to the manufacturer’s instructions. After amplification, 1 μl of product was added to a T7 and AsCas12a reaction master mix for RNA detection. This master mix contained 1× NEB 2.1 buffer, 62.5 nM AsCas12a, 112.5 nM ΨDNA (IDT), 500 nM FQ reporter (IDT), 1 mM rNTP mix (NEB, N0466), 2 U per ml of RNase inhibitor (NEB, M0314) and 12.5 U per ml of NxGen T7 RNA polymerase (Biosearch, 30221–1) in 20-μl reactions. Samples were then loaded into a 384-well plate, which was subsequently placed in a BioTek Synergy fluorescence microplate reader and incubated at 37 °C for 60 min. Fluorescence intensity readings for a FAM-labeled reporter were recorded at 483/20 nm (excitation) and 530/20 nm (emission) at 2.5-min intervals.

Amplicon sequencing for participant samples

Amplicon libraries were generated using SuperScript IV one-step RT–PCR followed by a second PCR with Q5 DNA polymerase to append Illumina barcodes. Libraries were pooled, gel-purified and sequenced on an Illumina MiSeqDx using a MiSeq Nano v2 kit. Sequencing reads were aligned to the HCV reference genome using Bowtie2 and SAMtools and visualized in JBrowse.

Mammalian cell culture

HEK293T (ATCC, CRL-3216), HeLa and MCF-7 cells were maintained in DMEM supplemented with 10% FBS and 1× penicillin–streptomycin at 37 °C with 5% CO₂. HepG2 cells were cultured in RPMI-1640 supplemented with 10% FBS and 1× penicillin–streptomycin under the same conditions. HeLa, MCF-7 and HepG2 cell lines were obtained from M. Xie (UF).

Mammalian cell transfection

Plasmids were cotransfected with ΨDNAs into HEK293T cells using TransIT-X2. Cells were seeded at 5 × 10⁵ cells per ml 48 h before transfection. For reporter assays, 200 ng of mCherry, 350 ng of AsCas12a–GFP (or GFP control) and 400 ng of ΨDNA were transfected in 48-well plates; ΨDNA was replaced with randomized DNA in control samples. For endogenous RNA knockdown, we used 350 ng of AsCas12a–GFP and 650 ng of ΨDNA (targeting or nontargeting) or ΨDNA alone (650 ng) in transduced cells. ΨDNA input was reduced to 175 ng in HeLa, MCF-7 and HepG2 cells and multiplex ΨDNAs were combined to 650 ng total. Cells were harvested at 16–24 h after transfection.

For RIP–qPCR and MeRIP–qPCR, 2 × 10⁶ HEK293T cells were transfected with 1.33 μg of plasmid and 1.07 μg of ΨDNA using 6 μl of TransIT-X2. For RNase H1–AsCas12a experiments, transfection conditions were identical to the reporter assay.

Flow cytometry for quantification of mCherry expression

At 16 h after transfection, cells were trypsinized (1× trypsin–EDTA), resuspended in FluoroBrite DMEM supplemented with 10% FBS and filtered through a 35-μm cell strainer. Samples were analyzed on a CytoFLEX LX flow cytometer. Fluorescence data were processed using FlowJo (version 10.10) and the MFI of mCherry was quantified in GFP-positive cells.

Compensation was performed using single-color controls (GFP, mCherry) and BrightComp eBeads. The gating strategy is shown in Supplementary Fig. 4.

RT–qPCR for relative quantification of mCherry and endogenous mRNA

Total RNA of samples was extracted using Monarch Total RNA miniprep kit (NEB, T2010S) as per the manufacturer’s instructions. Extracted RNA was later added to the RT–qPCR mix TaqMan fast virus one-step master mix (Thermo, 4444434). The reaction was performed on an Applied Biosciences QuantStudio 5 real-time PCR system and multiplexed with FAM probes for mCherry and endogenous genes and a Cy5 probe for GAPDH as the housekeeping gene. mCherry primers and probes were designed using the PrimeQuest tool from IDT and GAPDH primers and probes were from Asahi-Ozaki et al.⁴⁸. For endogenous genes (PPIA, RPL4, SMARCA4, NRAS and PCSK9), primers and probes were ordered from Thermo Fisher. The fold change was calculated relative to ΨNT using the ΔΔC_t method.

mRNA-seq for off-target effect quantification

HEK293T cells were seeded at 5 × 10⁵ cells per well in six-well plates 48 h before transfection. Plasmids (1 μg; AsCas12a–GFP, RfxCas13d–GFP, dCas13d–GFP or GFP only) were cotransfected with 2 μg of ΨDNA using 8 μl of TransIT-X2 in 250 μL Opti-MEM. After 18 h, cells were trypsinized and resuspended in PBS supplemented with 2% FBS and GFP-positive cells were sorted using a FACSymphony S6.

Total RNA was extracted from sorted cells using the Monarch Total RNA miniprep kit (NEB) and RNA quality was assessed before library preparation. mRNA libraries were prepared using the NEBNext Ultra II directional RNA library prep kit in combination with the NEBNext poly(A) mRNA magnetic isolation module. Libraries were indexed using NEBNext multiplex oligos and validated for size distribution and concentration before sequencing.

Sequencing was performed on an Illumina NovaSeq X Plus (100 cycles) or NextSeq 500 (150 cycles), generating ~30 million paired-end reads per sample. Reads were aligned using HISAT2, processed with SAMtools and quantified with featureCounts. Differential expression analysis was performed using DESeq2, and visualization (for example, volcano plots) was carried out in R. All computational analyses were conducted on the UF HiPerGator HPC system.

Lentiviral cell line production of AsCas12a–GFP-expressing HEK293T cells

Lentivirus was produced in HEK293T cells seeded at 7 × 10⁶ cells per 10-cm dish in 12 ml of medium (50% DMEM with 10% FBS and 50% Opti-MEM) 24 h before transfection. For transfection, 41 μl of Lipofectamine 3000 and 35 μl of P3000 were mixed with psPAX2 (1.3 pmol), pMD2.G (0.72 pmol) and AsCas12a–GFP transfer plasmid (1.64 pmol) in Opti-MEM, incubated for 20 min and added dropwise to cells. Medium was replaced 6 h after transfection. Viral supernatant was collected at 24 h and 52 h, pooled, clarified by centrifugation, filtered (0.45 μm) and concentrated 10× using a Lenti-X concentrator (Takara).

For infection, reverse transduction was performed by adding 15–150 μl of concentrated virus to 5 × 10⁴ HEK293T cells per well in six-well plates in the presence of 10 μg ml⁻¹ polybrene. After 72 h, cells were selected with 10 μg ml⁻¹ blasticidin. Cells were passaged no more than three times before downstream RNA knockdown experiments.

RIP–qPCR

RIP was performed as described previously⁴⁹. HEK293T cells transfected with HA–AsCas12a or HA–GFP and ΨPPIA were crosslinked with 1% paraformaldehyde for 15 min at room temperature and quenched with 125 mM glycine for 10 min. Cells were washed with cold PBS, harvested and lysed in buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT and 400 U per ml RNase inhibitor) for 20 min on ice. Lysates were clarified by centrifugation (15,000g, 15 min, 4 °C) and 30 μl was reserved as input.

Protein G agarose beads (20 μl per sample) were incubated with anti-HA antibody (2 μl) in wash buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT and 200 U per ml RNase inhibitor) for 2 h at 4 °C. Antibody-conjugated beads were then incubated with lysate overnight at 4 °C. Beads were washed three times with high-salt buffer (300 mM NaCl, 50 mM Tris-HCl pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT and 200 U per ml RNase inhibitor).

Bound complexes were eluted in SDS buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl pH 7.4) and treated with proteinase K (1.2 mg ml⁻¹) at 55 °C for 4 h. RNA was extracted using TRIzol, reverse-transcribed and quantified by RT–qPCR. Enrichment was calculated relative to input using the ΔΔC_t method.

MeRIP–qPCR

MeRIP–qPCR was performed with modifications to a previously described protocol. Briefly, 5 μg of total RNA was fragmented in buffer (50 mM Tris-HCl pH 8.0 and 50 mM MgCl₂) at 95 °C for 8 min and 10% was reserved as input. For IP, 30 μl of protein G beads were washed and incubated with anti-m⁶A antibody (3 μl) overnight at 4 °C in IP buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.5 and 0.1% NP-40). Antibody-conjugated beads were then incubated with 4.5 μg of fragmented RNA in IP buffer supplemented with RNase inhibitor for 4 h at 4 °C.

Beads were washed five times with IP buffer and bound RNA was released by proteinase K digestion (5 μl) at 37 °C for 60 min followed by 56 °C for 15 min. RNA from input and IP samples was extracted using TRIzol, reverse-transcribed and quantified by qPCR using TaqMan assays. Enrichment was calculated relative to input using the ΔΔC_t method.

Ribo-seq for ribosomal profiling

HEK293T cells were seeded at 5 × 10⁵ cells per well in six-well plates 48 h before transfection. Cells were transfected with 1 μg of AsCas12a–GFP and 2 μg ΨDNA using 8 μl of TransIT-X2 in 250 μl of Opti-MEM per well. After 15 h, cells were harvested, pooled in six wells per condition as one biological replicate (n = 2) and subjected to GFP sorting (~5 × 10⁶ GFP⁺ cells per sample).

Following sorting, 20% of cells were used for total RNA extraction and mRNA-seq library preparation as described above. The remaining 80% were used for ribosome profiling using the All-In-One RiboLace gel-free kit (IMMAGINA) as per the manufacturer’s instructions.

Sequencing was performed on an Illumina NovaSeq X Plus (1× 100 bp), generating ~300 million reads per sample. Reads were processed by bcl-convert and fastp, trimmed with cutadapt (minimum length: 29 nt) and unique molecular identifiers were extracted using UMI-tools. Ribosomal and transfer RNA reads were removed using Bowtie2 and the remaining reads were aligned to the hg38 genome using STAR.

Ribosome profiling quality control was performed using RiboWaltz with a P-site offset of 12 nt. Gene-level counts from mRNA-seq and Ribo-seq were obtained using featureCounts and differential translation efficiency was analyzed using RiboDiff. Results were visualized using R.

CLIP-seq

CLIP-seq was performed with minor modifications to a previously described protocol^50,51. HEK293T cells stably expressing AsCas12a (1.5 × 10⁷ cells per sample) were labeled with 4-thiouridine (200 μM) and ultraviolet-crosslinked twice at 365 nm (150 mJ cm⁻²). Cells were lysed and partially digested with RNase T1 (0.2 U per μl). Clarified lysates were subjected to IP using Dynabeads protein G preconjugated with anti-AsCas12a antibody (10 μg of antibody per 100 μl of beads per 1 mg of lysate).

Following IP, complexes were washed and subjected to a second RNase T1 digestion (10 U per μl, 10 min, 22 °C), followed by high-salt washes. RNA–protein complexes were treated with proteinase K (37 °C, 15 min) and RNA was extracted by phenol–chloroform.

cDNA libraries were prepared using the NEBNext Ultra II directional RNA library prep kit and sequenced on an Illumina NovaSeq platform (2× 150 bp), generating ~50 million reads per sample.

Dual RNA–DNA editing with AsCas12a

HEK293T cells were seeded at 5 × 10⁵ cells per ml 48 h before transfection. Cells were transfected with 350 ng of AsCas12a–GFP or GFP-only plasmid and either 650 ng of ΨNT or a combination of 325 ng of ΨPPIA/RPL4 and 325 ng crCCR5 using 2 μL TransIT-X2 in 50 μl of Opti-MEM. Complexes were incubated for 25 min and added to 48-well plates.

At 18 h after transfection, cells were harvested, washed and divided into three fractions. One fraction was used for RNA extraction, one was used for immediate DNA extraction using QuickExtract solution (30 μl) and the third was reseeded for DNA extraction at 50 h after transfection. RNA was analyzed by qPCR and mRNA-seq and DNA was subjected to amplicon sequencing. Indels were quantified using CRISPResso2.

PLA

HEK293T cells were seeded at 5 × 10⁵ cells per ml in chamber slides (Lab-Tek) 48 h before transfection. Cells were transfected with 350 ng of AsCas12a–GFP or dRfxCas13d–GFP and 650 ng of ΨPPIA using 2 μl of TransIT-X2 in 50 μl of Opti-MEM. After 12 h, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature, followed by permeabilization with 0.5% Triton X-100 for 10 min.

The PLA was performed using the Duolink in situ red kit according to the manufacturer’s instructions. Primary antibodies included anti-HA (for AsCas12a or dRfxCas13d) and anti-RNase H1, incubated overnight at 4 °C. Imaging was performed using a Leica fluorescence microscope.

Target design

Target sites were designed on the basis of RNA secondary structure and RBP-binding information. SHAPE reactivity scores from RASP version 2.0 were used to identify structurally accessible regions^30–32 and RBP-binding sites were obtained from POSTAR3 to guide target selection³³ from the available datasets.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Extended Data

Extended Data Fig. 3 | — (a-b) Binding affinity (Kd) values for different guide constructs obtained via Bio-Layer Interferometry (BLI). This includes crRNA, no-scaffold guide RNA, cDNA, and cDNA with 5’ and 3’ handles (ΨDNA). Log10 concentration plotted on right pannels. (c) Table of binding kinetic parameters derived from BLI. Kd is reported with steady-state measurements (left) obtained from response curves in (a), and kinetic measurements (right) accounting for the reported ratios between kon and koff. (d-e) Michaelis-Menten analysis reveals robust trans-cleavage activity with a with ΨDNA-ssRNA complexes. Fluorescence time courses at indicated substrate concentrations are shown (lines, mean; shaded areas, SD; n = 3 technical replicates). Representative plots of initial velocity versus time for ΨDNA and ssRNA target. Using 1.25 nM effective AsCas12a-ΨDNA-RNA complex and increasing fluorophore–quencher (F–Q) substrate concentrations at 37 °C. Calculated kcat, Km, kcat/Km and (α, β, γ) values report the mean ± SD (n = 3).

Extended Data Fig. 4 | — **(a)** Detection of mimics of endogenous mRNAs by AsCas12a and ΨDNA. Fluorescence measurements for mRNA detection with ΨDNA and AsCas12a. Endogenous mRNAs from HEK293T cells were reverse-transcribed, amplified by RT–PCR, and subsequently transcribed in vitro. A total of 18 mRNA targets were tested. Fold change of fluorescence intensity normalized to No Target Control (NTC). (b) Heatmap displaying the tolerance of AsCas12a to different direct repeat sequences derived from diverse Cas12 systems. The heatmap illustrates the relative fluorescence intensity, indicating the detection efficiency. Some non-canonical scaffold sequences enhance the detection activity compared to their canonical scaffolds, suggesting potential for further optimization of ΨDNA guides (n = 3). **(c-e)** Sensitivity analysis of RNA detection using different scaffold sequences with AsCas12a enzyme. Ratio of RFU at t = 60 min to RFU at t = 0 min at different target RNA concentrations for **(c)** AsCas12a, **(d)** LbCas12a, and **(e)** OpCas12b scaffold sequences. Threshold set at mean No Target Control (NTC) plus three times the standard deviation (σ). **(f)** AsCas12a and **(g)** Cas12i1 trans-cleavage activity with crRNAs of different scaffolds to detect an ssDNA target. The scaffolds tested were selected based on their high trans-cleavage activity when used as 3′ handles in ΨDNA-mediated RNA detection. Sequence alignments of HCV patient samples obtained by NGS of **(h)** the 5′UTR and **(i)** E2 regions. **(h-i)** Gray sections represent matched bases to the reference genome and colored sections represent mismatched bases. Alignments were visualized by JBrowse. **(a, c-g)**Error bars represent mean +/− SD (n = 3).

Extended Data Fig. 5 | — **(a)** Schematic representation of ΨPCSK9 screening to mediate mRNA knockdown. Relative mRNA expression levels of PCSK9 transcripts in cells transfected with five different ΨDNAs compared to a non-targeting (NT) guide. **(b-d)** Influence of RNA secondary structure on AsCas12a-ΨDNA-mediated knockdown. RNA knockdown evaluated with relative quantification of different ΨDNAs in PPIA, RPL4 and PCSK9 transcripts. All RNA expression levels are normalized with their non-target ΨDNA. Colored dots are groups transfected with ΨDNAs tested in Fig. 3h. SHAPE scores were obtained from the RASP v2.0 database. The plots show no influence of RNA secondary structure on AsCas12a–ΨDNA–mediated knockdown. Data points without visible error bars indicate that the SEM values are too small to be displayed in GraphPad Prism. **(b)** Knockdown of PPIA transcripts. **(c)** Knockdown of RPL4 transcripts. **(d)** Knockdown of PCSK9 transcripts. **(e-f)** Influence of RBP binding on AsCas12a-ΨDNA-mediated knockdown. RNA knockdown evaluated with relative quantification of PPIA and RPL4 mRNA with non-targeting ΨDNA (gray) or gene-specific ΨDNAs (colored). The plots show that RBP binding inhibits AsCas12a–ΨDNA–mediated knockdown of PPIA transcripts but not RPL4. Statistical analyses were performed using one-way ANOVA (two-tailed, α = 0.05). **(e)** Knockdown of PPIA transcripts. **(f)** Knockdown of RPL4 transcripts. **(g)** Relative quantification of PPIA expression. Comparing dAsCas12a (gray) and AsCas12a (blue) mediated knockdown with non-target ΨDNA or ΨPPIA. No significant differences were detected. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test (two-tailed, α = 0.05). **(h)** Comparison among GFP control, AsCas12a with ΨDNA, and RfxCas13d with crRNA. Relative quantification of PPIA and RPL4 expression, all groups are normalized by NT. **(a-h)** Bars represent the mean value +/− standard error of mean (SEM) of (n = 3) biological replicates.

Extended Data Fig. 6 | — **(a-b)** Volcano plots from mRNA-Seq data comparing the off-target effects of AsCas12a-ΨDNA for **(a)** PPIA and **(b)** RPL4 gene. Each set illustrates the off-target effects of AsCas12a–ΨDNA–mediated RNA knockdown of AsCas12a-ΨDNA RNA knockdown when compared to AsCas12a non-target-ΨDNA or dCas13d non-targeting crRNA control to evaluate gene expression differences among different samples. Plots and statistical analyses were in DESeq2 with 95% CI with n = 2 biologically independent replicates with 30 million reads each. All colored points (blue for PPIA and green for RPL4) represent genes that show statistically significant in downregulation (left) or upregulation (right) with gene of interest labeled. Exact quantification and comparison are displayed to evaluate the difference in off-target effects among each different control. DESeq2 (negative binomial model) with Wald test and Benjamini–Hochberg procedure adjustment; volcano plot shows log2 fold change vs −log10 FDR. **(c-d)** Gene expression in counts per million of target genes (PPIA and RPL4) and other specified genes that have similar sequences to evaluate mismatch specificity for AsCas12a-ΨDNA, ΨDNA-only, and Cas13d-crRNA constructs. This analysis was performed based on the approach described by H. Tong et al.⁵². **(e)** Ribo-Seq quality control of trinucleotide periodicity for ΨNT and ΨPPIA. Above 60% percent of filtered reads have no codon shift. Data obtained with Ribowaltz (biological replicates, n = 2). **(f)** Ribo-Seq quality control of coding sequences (CDS) reads for ΨNT and ΨPPIA. This demonstrates experimental procedure successfully captures active ribosomes on coding regions. Above 90% percent of filtered reads are in coding regions. Data obtained with Ribowaltz (biological replicates, n = 2). Right panel portrays the distribution of total RNA without ribosomal enrichment. **(g)** Reads coverage of CLIP-seq data. Ψ-NT represents non-target ΨDNA; ΨPPIA represents ΨDNA target PPIA transcripts; Ψ-MIX represents mix of 4 different ΨDNAs (ΨPPIA, ΨRPL4, ΨSMARCA4 and ΨPCSK9). Read counts were normalized to the total number of reads. Red arrow represents ΨDNA position. Bigwig files are visualized by IGV. Read coverage shows that AsCas12a specifically captures target transcripts guided by ΨDNA.

Extended Data Fig. 7 | — **(a)** Endogenous mRNA knockdown induced by AsCas12a and ΨDNA in different cell lines. Relative quantification of PPIA mRNA in HeLa, HepG2, and MCF-7 cells expressing either GFP (gray, control) or AsCas12a (blue) with non-targeting ΨDNA (NT) or gene-specific ΨDNA (ΨPPIA). Statistical analyses were performed using two-way ANOVA followed by Sidak’s multiple comparisons test (two-tailed, α = 0.05). **(b)** Relative quantification of RPL4 mRNA. Comparison between full-length RNaseH1 (RNaseH1-FL, gray) and the C-terminal domain of RNaseH1 (RNaseH1-C, green). **(c)** Microscopy images for cells treated with RNaseH-AsCas12a-GFP, mCherry, and two different conditions of ΨDNA (ΨNT and ΨmCherry). Individual GFP and mCherry channels are shown as well as the overlay. Fluorescence microscopy was performed in 3 independent replicates, all showing consistent results. **(d)** Relative mRNA expression for co-transfection of RNase H-AsCas12a-GFP, AsCas12a-GFP or GFP with mCherry and targeting and non-targeting ΨDNA to induce RNA knockdown. Relative quantification of mCherry mRNA in HEK293T cells shows that RNase H–fused AsCas12a induces higher RNA knockdown compared to wild-type AsCas12a. Statistical analysis was performed using two-way ANOVA (two-tailed, α = 0.05). **(e)** Multiplex knockdown of four endogenous transcripts, PPIA, RPL4, NRAS, and SMARCA4 using non-targeting ΨDNA (ΨNT) or various ΨDNA combinations as shown. Results highlight the potential for multiplex RNA targeting using the ΨDNA-guided CRISPR-Cas12a platform. **(a, b, d, e)** Error bars represent the mean +/− SEM (n = 3).

Supplementary Material

Supplementary Data 1 [Protein sequences, oligo sequences, RNA secondary-structure analysis data and ΨDNA library (Supplementary Tables 1–4)]

NIHMS2179613-supplement-Supplementary_Data_1__Protein_sequences__oligo_sequences__RNA_secondary-structure_analysis_data_and__DNA_library__Supplementary_Tables_1_4__.xlsx^{(48.8KB, xlsx)}

Reporting Summary

NIHMS2179613-supplement-Reporting_Summary.pdf^{(1.7MB, pdf)}

Source Data

NIHMS2179613-supplement-Source_Data.pdf^{(2.3MB, pdf)}

Supplementary Information

NIHMS2179613-supplement-Supplementary_Information.pdf^{(2.1MB, pdf)}

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41587-026-03129-w.

Acknowledgements

We extend our gratitude to the HCV-TARGET consortium, particularly D. Nelson and L. Morelli, for their expert guidance and provision of clinical samples. We also thank the members of the P.K.J. and G. P. Wang labs for valuable discussions and UF Health Cancer Center for their support; in particular, we thank L. Mourad, M. Ahmadimashhadi and Z. Fang for their help with experiments. Lastly, we thank C. Denard and his lab members in the Department of Chemical Engineering, B. Osborn from GatorBio and the UF Interdisciplinary Center for Biotechnology Research (ICBR) Monoclonal Antibody Core (RRID:SCR_019147) for their guidance on BLI experiments, the UF ICBR Cytometry Core (RRID:SCR_019119) for their help with flow cytometry experiments and the UF ICBR Next-Gen DNA Sequencing Core (RRID:SCR_019152) for their help with NGS in the NovaSeq X Plus. HeLa, HepG2 and MCF-7 cell lines were kindly provided by M. Xie at UF. This work was funded by UF, the UF Herbert Wertheim College of Engineering, the Shah Foundation Endowment Funds (P.K.J.), Exxon Mobil Gator Alumni Faculty Endowed Funds (P.K.J.), the National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (R21AI156321, R21AI168795 and R61AI181016 to P.K.J.) and National Institute of General Medical Sciences (R35GM147788 to P.K.J.) and scholarships from UF (I.B. and A.F.). The funding sources had no role in study design, data collection, analysis and interpretation or manuscript preparation.

Footnotes

Online content

Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41587-026-03129-w.

Competing interests

C.O., B.H., S.R.R. and P.K.J., are listed as inventors on a patent application related to the content of this work. P.K.J. and S.R.R. are cofounders of CasNx, LLC and P.K.J. is a cofounder of CRISPR, LLC. The remaining authors declare no competing interests.

Extended data is available for this paper at https://doi.org/10.1038/s41587-026-03129-w.

Peer review information Nature Biotechnology thanks Chase Beisel for their contribution to the peer review of this work.

Data availability

The data supporting the findings of this study are available within the article and its Supplementary Information. Raw sequencing data were deposited to the National Center for Biotechnology Information Sequence Read Archive under BioProjects PRJNA1401894 and PRJNA1274432 and are publicly available. Additional materials are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Associated Data

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

Supplementary Materials

Supplementary Data 1 [Protein sequences, oligo sequences, RNA secondary-structure analysis data and ΨDNA library (Supplementary Tables 1–4)]

NIHMS2179613-supplement-Supplementary_Data_1__Protein_sequences__oligo_sequences__RNA_secondary-structure_analysis_data_and__DNA_library__Supplementary_Tables_1_4__.xlsx^{(48.8KB, xlsx)}

Reporting Summary

NIHMS2179613-supplement-Reporting_Summary.pdf^{(1.7MB, pdf)}

Source Data

NIHMS2179613-supplement-Source_Data.pdf^{(2.3MB, pdf)}

Supplementary Information

NIHMS2179613-supplement-Supplementary_Information.pdf^{(2.1MB, pdf)}

Data Availability Statement

[R1] 1.Jinek M et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R2] 2.Cong L et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] 3.Gootenberg JS et al. Nucleic acid detection with CRISPR–Cas13a/C2c2. Science 356, 438–442 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Chen JS et al. CRISPR–Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436–439 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Zetsche B et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759–771 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] 6.Abudayyeh OO et al. RNA targeting with CRISPR–Cas13. Nature 550, 280–284 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R7] 7.Konermann S et al. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors. Cell 173, 665–676 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] 8.Chen J et al. Trans-nuclease activity of Cas9 activated by DNA or RNA target binding. Nat. Biotechnol 43, 558–568 (2025). [DOI] [PubMed] [Google Scholar]

[R9] 9.Teng F et al. CDetection: CRISPR–Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity. Genome Biol. 20, 1–7 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Stella S, Alcón P & Montoya G Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage. Nature 546, 559–563 (2017). [DOI] [PubMed] [Google Scholar]

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PERMALINK

DNA-guided CRISPR–Cas12 for cellular RNA targeting

Carlos Orosco

Boyu Huang

Santosh R Rananaware

August P Bodin

Ivy Browning

Anne Fang

Michael P Baugh

Ian H Lange

Yasmin B Elhabashy

Mahika Balaraju

Jordan G Lewis

Nayan H Shah

Michael P Hanna

Sarah J Flannery

Katelyn S Meister

Vedant Karalkar

Piyush K Jain

Abstract

Results

Engineering of ΨDNAs for RNA detection using Cas12 enzymes

Fig. 1 |. Engineering ΨDNA guides for RNA detection using Cas12 enzymes.

Translational repression in HEK293T cells with ΨDNA and AsCas12a

Fig. 2 |. Translational repression of mCherry with ΨDNA–AsCas12a complex in HEK293T cells.

Efficient endogenous RNA knockdown using ΨDNA-guided CRISPR–Cas12a with low off-target effects

Fig. 3 |. mRNA targeting of endogenous genes using ΨDNA–AsCas12a, off-target effect evaluation and integrated RNA-binding and translation profiling.

Potential application of AsCas12a and ΨDNA system

Fig. 4 |. Programmable applications of the ΨDNA–AsCas12a system.

Discussion

Methods

Plasmid construction

Protein expression and purification

Oligonucleotide preparation

CRISPR–Cas-based fluorescence detection assay

EMSA

Biolayer interferometry

Systematic ΨDNA evaluation assay

IVT

Endogenous mRNA detection

Participant sample collection

Participant sample extraction

Participant sample validation

Amplicon sequencing for participant samples

Mammalian cell culture

Mammalian cell transfection

Flow cytometry for quantification of mCherry expression

RT–qPCR for relative quantification of mCherry and endogenous mRNA

mRNA-seq for off-target effect quantification

Lentiviral cell line production of AsCas12a–GFP-expressing HEK293T cells

RIP–qPCR

MeRIP–qPCR

Ribo-seq for ribosomal profiling

CLIP-seq

Dual RNA–DNA editing with AsCas12a

PLA

Target design

Reporting summary

Extended Data

Extended Data Fig. 1 |. Characterization of ΨDNA-Cas12 trans-cleavage for RNA detection.

Extended Data Fig. 2 |. Binding and efficiency of RNA cleavage by ΨDNA-Cas12 complexes.

Extended Data Fig. 3 |. Enzyme kinetics of AsCas12a-ΨDNA-RNA complex.

Extended Data Fig. 4 |. Targeting of long RNAs with ΨDNA-Cas12 and scaffold optimization for limit-of-detection analysis.

Extended Data Fig. 5 |. Characterization of ΨDNA screening across multiple targets and Cas effectors.

Extended Data Fig. 6 |. Specificity of the AsCas12a–ΨDNA system assessed by RNA-seq, Ribo-seq, and CLIP-seq.

Extended Data Fig. 7 |. Applications of the AsCas12a-ΨDNA system.

Supplementary Material

Acknowledgements

Footnotes

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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