Introduction

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) technology has transformed our approach to exploring and modifying biological systems, offering a powerful tool for nucleic acid targeting and cleavage that enables rapid identification, editing, and manipulation of genetic material1,2. Cas12a is an extensively studied Class 2/Type V CRISPR-Cas endonuclease. It binds to and cleaves double-stranded DNA (dsDNA) in a sequence-specific manner guided by CRISPR RNA (crRNA), a process referred to as cis-cleavage3. This is followed by the activation of trans-cleavage, enabling Cas12a to indiscriminately cleave surrounding single-stranded DNA (ssDNA)4. Cas12a features T-rich targeting3, high specificity5, short crRNA for easy processing and synthesis6, along with characteristic trans-cleavage activity4, making it widely applicable in gene editing and nucleic acid diagnostics3,4,7,8,9.

Regulating the complex functions of Cas12a is crucial for its effective use. Both enhancing and suppressing its nuclease activity have valuable applications. Increased activity usually enables more efficient gene editing in eukaryotic cells. Prominent examples include two engineered Cas12a variants, hyperCas12a (D156R/D235R/E292R/D350R) and enAsCas12a (E174R/S542R/K548R), both of which show impressive performance in indel introduction, expression regulation, epigenetic modifications, and base editing8,10. In the context of CRISPR-based nucleic acid diagnostics, the enhanced activity of enAsCas12a enables it to tolerate temperatures up to 65 °C, making it compatible with the loop-mediated isothermal amplification (LAMP)11. Conversely, in prokaryotic genome editing, reduced cis-cleavage activity can alleviate toxicity, improving transformation efficiency and editing efficiency12,13. For nucleic acid diagnostics, lowering target DNA cleavage helps balance between nucleic acid amplification with CRISPR detection, thereby enhancing the sensitivity of one-pot assays14,15. These examples highlight the importance of tailoring Cas12a activity to application-specific requirements in both gene editing and nucleic acid diagnostics. Achieving this necessitates a simple and flexible strategy for regulating Cas12a activity.

To achieve effective activity regulation, existing strategies primarily involve modifying CRISPR components. Some researchers have focused on protein engineering to develop enhanced mutants, such as hyperCas12a and enAsCas12a mentioned above, which demonstrate improved performance in expression regulation, genome editing, and nucleic acid diagnostics8,10,11. Others have concentrated on crRNA modifications, employing strategies such as adding a 7-mer DNA extension, incorporating 2-aminoadenine (base Z), or truncating/splitting the crRNA16,17,18,19,20. These approaches have succeeded in regulating Cas12a’s nuclease activity or improving the specificity, and are generally used in in vitro nucleic acid diagnostics. While substantial progress has been made in Cas12a engineering and crRNA modifications, challenges still exist to enhance both the flexibility and simplicity of current systems. From a flexibility standpoint, the targeted modification of Cas12a or crRNA results in powerful mutants with limited versatility, thus constraining their deployment to specific contexts (Table 1). As an illustration, split crRNA strategies are challenging to implement in vivo, whereas circular crRNAs offer enhanced stability in cellular contexts but may have limited utility in in vitro diagnostic applications18,21. Regarding simplicity, both the in vivo construction and in vitro purification of Cas12a mutants impose considerable workload and cost. Similarly, the synthesis of artificially modified crRNAs—such as those incorporating 2-aminoadenine or extended with DNA segments—remains limited by current oligonucleotide synthesis technologies, rendering the process both expensive and time-consuming (Table 1)16,17. Therefore, there remains a need for a flexible and simple strategy for the efficient and stable regulation of Cas12a activity to promote its applications.

Table 1 Comparison among Cas12a crRNA engineering strategies
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Here, taking LbCas12a derived from Lachnospiraceae bacterium ND2006 as an example, we systematically explore the impact of crRNA direct repeat (DR) sequence mutations on Cas12a activity. Leveraging the ease of construction and inherent diversity of DR sequence mutants, we achieve flexible regulation of Cas12a activity. We construct a mutant toolbox through high-throughput screening and validate it via in vitro characterization that these mutants endow distinct properties to Cas12a. Exploiting the complementarity and synergy between crRNA mutants, we demonstrate the practical applicability of this toolbox in different application scenarios. Specifically, we address issues of leaky repression in CRISPR interference (CRISPRi) by providing a finely tunable expression suppression strategy, achieving greater precision in base editing, enhancing the homologous recombination-mediated gene editing efficiency of LbCas12a in prokaryotes, and resolving the inherent limitations of CRISPR-Dx for rapid, simple, and accurate one-pot and quantitative analysis. Overall, our DR mutation strategy showcases its powerful potential, significantly expanding the existing Cas12a toolbox.

Results

Construction and high-throughput screening of crRNA DR library

The DR of Cas12a crRNA features a short stem-loop-like structure that is crucial for its stability and Cas12a recognition (Fig. 1a)22. While utilizing crRNA to guide DNase-dead Cas12a (dCas12a, D832A)23 for to repress the expression of green fluorescence protein (GFP), we unexpectedly observed that mutations in the crRNA flanking sequence led to variations in fluorescence expression levels (Fig. 1b and Supplementary Fig. 1, 2). This observation suggests that Cas12a activity can be effectively modulated through targeted alterations in the DR sequence. Such mutations likely influence RNA conformation, perturb the Cas12a-crRNA interaction, and consequently affect the overall functionality of the Cas12a-crRNA ribonucleoprotein (RNP). To explore this possibility, we divided the DR sequence into three parts—stem, loop, and flanking sequence—and constructed saturated mutant libraries in each part separately.

Fig. 1: Construction and screening of crRNA DR library.
figure 1

a Schematic of the secondary structure of canonical crRNA. The DR sequence is divided into three parts: flanking sequence, loop and stem, with each region mutated separately. b Fluorescence intensity derived from flanking sequence-mutated crRNAs guiding dCas12a to knock down gfp. c Screening of the crRNA DR library. crRNA mutants were used to guide dCas12a to knock down gfp and ranked based on the level of expression repression. d Relative expression levels of gfp derived from single-part-mutated crRNAs. The eight mutants in the black box are derived from Fig. 1b.e Relative expression levels of gfp derived from the multi-part-mutated crRNAs. The flanking sequences of the multi-part mutants were CAAUG or GAAUG, with the loop contained the mutation library. f Typical crRNA mutant sequences determined through Sanger sequencing and their corresponding designations. The left column indicates their position in (e) and (f), while the right column contains the designations used for referencing these crRNAs. g Diversity of gfp relative expression levels derived from the diversified crRNA mutants. The relative expression was calculated based on the specific fluorescence level (RFU/OD600) and normalized by setting the value of the non-targeted control as 100%. Source data are provided as a Source Data file.

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For high-throughput characterization of the activities of these crRNA mutants, we developed a CRISPRi system where the dCas12a was guided by crRNA mutants to knock down gfp (Fig. 1c). The crRNA activities exhibited diversity, as indicated by the varied relative expression level, preliminarily confirming that DR mutation is an effective approach to obtain easily regulated RNP with diversified activity (Fig. 1d). Under all three mutation conditions, the majority of the loop-mutated crRNAs showed relatively strong repression of the expression of GFP, suggesting the retention of their functional activities. The 96 screened mutants exhibited a wide dynamic range of gfp relative expression, ranging from 31.8 to 110.1% (Fig. 1d). Of these, 80 mutants showed expression levels below 70% (Fig. 1d). In contrast, flanking sequence-mutated crRNAs as well as stem-mutated crRNAs were more likely to lose function, possibly due to excessive disruption of the canonical structure (Fig. 1d). The mutants exhibited a relatively narrow dynamic range in regulating gfp expression. For the 48 flanking sequence mutants and 48 stem mutants, the gfp relative expression levels ranged from 50.1 to 100.7% and from 47.8 to 101.4%, respectively (Fig. 1d). Within each group, only 18 and 27 mutants showed expression levels below 70% (Fig. 1d). We determined the sequences of 31 single-part-mutated crRNAs with the various inhibition effect on gfp through sanger sequencing (Supplementary Fig. 2). LbCas12a appeared to have a high tolerance for mutations in crRNA DR sequence, particularly in the loop, where all the positions tolerated all four bases-A, G, C, and U. This indicates that LbCas12a has a diverse selection of crRNA sequences, which could offer great potential for activity regulation.

Further, we explored the potential of introducing mutations across multiple parts of the DR sequence. Given that stem mutations tend to cause functional loss of crRNA due to structural disruptions, we focused on introducing multiple mutations in the flanking sequence and the loop. We selected the two flanking sequence-mutated crRNAs that exhibited the strongest gfp repression and constructed saturated mutant libraries in their loops (Fig. 1e). These multi-part-mutated crRNAs were more prone to lose activity. Among the 96 screened mutants, GFP relative expression levels ranged from 49.7 to 112.5% (Fig. 1e). Only 13 mutants demonstrated appreciable activity, lowering GFP expression to below 70% (Fig. 1e). We identified the sequences of the 20 multi-part-mutated crRNAs that exhibited the strongest gfp repression through Sanger sequencing (Supplementary Fig. 3). Notably, the mutations in loop mutant D22 can be added to flanking sequence mutant A3 and D5. The resulting multi-part mutant (C39 and A46) still exhibited adequate activity.

Among the 288 mutants we screened, we selected 6 exhibiting distinct inhibitory activities, together with the canonical crRNA, to form a “crRNA toolbox” (Fig. 1f). The two flanking sequence mutants were designated as “F1 (Flanking sequence-1)” and “F2 (Flanking sequence-2)”, the two loop mutants as “L1 (Loop-1)” and “L2 (Loop-2)”, and the two multi-parts mutants as “FL1 (Flanking sequence-Loop-1)” and “FL2 (Flanking sequence-Loop-2)”. These mutants were functional as CRISPRi components. We took them as examples to further explore how DR mutations affected the activity of Cas12a-crRNA RNP, and whether these mutated RNPs could be applied in other scenarios, both in vitro and in vivo. Nevertheless, beyond these six sequences, the DR mutation strategy generated a large and highly diverse set of crRNA sequences that, as CRISPRi components, demonstrated varying levels of expression repression (Fig. 1g). These sequences could serve as a valuable backup arsenal, offering greater possibilities for fine-tuning Cas12a activity through DR mutations.

Tunable activity of Cas12a guided by crRNA toolbox

To investigate how DR mutations influence the characteristics of the Cas12a-crRNA RNP, we initially examined the interaction between crRNA mutants and Cas12a, as the formation of the binary complex is fundamental to the subsequent functions of the RNP. We assessed the affinity between Cas12a and the crRNAs using surface plasmon resonance (SPR) analysis (Supplementary Fig. 4). Although slight variations were observed, the differences remained within a twofold range, indicating relatively similar binding affinities (Supplementary Fig. 4). These results suggest that LbCas12a possesses a relatively high tolerance to DR region mutations in crRNA with respect to binding. The observed differences in binding affinity did not correlate with the functional performance of these crRNAs in downstream assays such as cis- and trans-cleavage. This indicates that while Cas12a–crRNA binding is necessary, it is not the primary determinant of Cas12a-crRNA activity.

We subsequently characterized the affinity of the dCas12a for target dsDNA after forming RNP complexes with the crRNAs using electrophoretic mobility shift assay (EMSA). The canonical crRNA demonstrated a superior ability to guide dCas12a in binding to target dsDNA compared to the crRNA mutants (Fig. 2a). Among the six mutants, F1 and F2 exhibited the highest affinity for target dsDNA, followed by L1 and L2, while FL1 and FL2 showed the weakest binding (Fig. 2a). These findings indicate that dCas12a acquires diverse dsDNA binding affinities when paired with different crRNAs, aligning with our observations from screening the crRNA library using CRISPRi. This variability may further contribute to differences in cis- and trans-cleavage activities.

Fig. 2: In vitro characterization of crRNA mutants.
figure 2

a The binding affinity of RNPs for dsDNA. dCas12a-crRNA RNPs (0, 30, 60, 90, 120, and 150) were incubated with 5 nM dsDNA at 37 °C for 15 min, and EMSA were performed to determine the bound and unbound dsDNA. b Fluorescent characterization of cis-cleavage. The FAM- and BHQ1-labeled dsDNA probe was cleaved by Cas12a-crRNA RNP to generate fluorescence. Shaded areas around the curves represent the standard deviation (SD) of three independent experiments. c Fluorescent characterization of trans-cleavage at various target dsDNA substrate (10 nM, 1 pM, 10 pM), with 8 C ssDNA probe cleaved to generate fluorescence. Fluorescence intensity at 40 min with background subtracted is shown. d Fluorescence signals from trans-cleavage of RNPs activated by target dsDNA containing double-point mutations. The upper panel shows the sequence of the mutated targets. The lower panel presents the fluorescence signal at 40 min for each crRNA, normalized to the perfect match signal after background subtraction. MM indicates mismatches. Data are shown as mean ± s.d. for n  =  3 biologically independent samples. Source data are provided as a Source Data file.

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To assess how crRNA toolbox affects the cis-cleavage activity of Cas12a, we employed a target dsDNA reporter, composed of a 3’-FAM-labeled non-target strand (NTS) and a 5’-BHQ1-labeled target strand (TS), which denatures and displays increased fluorescence upon cis-cleavage (Fig. 2b and Supplementary Fig. 5a). We initially performed the cis-cleavage assay using Buffer-1, a simple, laboratory-prepared solution identical to that used in EMSA (Fig. 2a). Under this condition, Cas12a guided by FL1 and L1 failed to produce any detectable cis-cleavage signals, while the remaining crRNAs exhibited activity in the following order: Canonical > F2 > F1 > L2 > FL2 (Fig. 2b). Following binding to target dsDNA, the cis-cleavage activity of Cas12a guided by crRNAs in Buffer-1 generally corresponded to their binding affinity to target dsDNA. Exceptions were noted with FL2 and L1. While FL2 exhibited slightly weaker binding affinity to target dsDNA compared to L1, its cis-cleavage activity was higher. This suggests that DR mutations may impact Cas12a’s cis-cleavage activity not only through altering substrate dsDNA affinity, but also by directly affecting the nuclease activity itself.

To see whether L1 and FL1 could mediate cis-cleavage, we repeated the cis-cleavage assays using a more complex, commercially available rCutSmart Buffer. In this more optimal Buffer, differences among the crRNAs became less pronounced. Cas12a guided by the canonical crRNA and F2 demonstrated the highest levels of cis-cleavage activity within the crRNA toolbox (Supplementary Fig. 5b). The cis-cleavage activities of Cas12a guided by L2, FL2, and F1 were similar, demonstrating moderate efficiency (Supplementary Fig. 5b). In contrast, Cas12a guided by FL1 showed lower cleavage activity than the above mentioned crRNAs, with L1-guided Cas12a exhibiting the lowest cleavage activity overall (Supplementary Fig. 5b). These results indicate that the crRNA toolbox exhibits diversity in guiding Cas12a for cis-cleavage, demonstrating that the cis-cleavage activity of Cas12a can be readily regulated by altering the crRNA sequence.

To investigate how crRNA toolbox affects the trans-cleavage activity of Cas12a, we used an ssDNA reporter composed of a FAM and a BHQ1 linked by a CCCCCCCC sequence (hereafter referred to as 8 C ssDNA probe), which shows increased fluorescence upon cleavage (Fig. 2c and Supplementary Fig. 6a). At a relatively high concentration of target dsDNA (10 nM), the crRNAs produced comparable trans-cleavage signals except L1, which showed the lowest activity in guiding Cas12a for trans-cleavage (Fig. 2c and Supplementary Fig. 6b). However, as the concentration of target DNA decreased, the crRNA toolbox showcased its diversity (Fig. 2c and Supplementary Fig. 6c, d). Consistent with the cis-cleavage results, Cas12a guided by the canonical crRNA still exhibited the strongest activity, followed closely by F2. L2 and F1 displayed similar levels of activity, forming the next tier. Further down were FL1 and FL2, which performed slightly better than L1. This order was generally consistent with the performance of crRNAs in dsDNA binding and cis-cleavage, which is expected, as trans-cleavage is a subsequent step following these processes. As the final step in Cas12a activity, trans-cleavage activity can also be regulated by substituting the DR sequence.

Next, we characterize the specificity of crRNA toolbox in discriminating point-mutations across target dsDNA. We employed dsDNA activators with two consecutive mutations at each position across the protospacer and measured the trans-cleavage fluorescence signal of Cas12a guided by members of the crRNA toolbox (Fig. 2d). The specificity of Cas12a guided by each mutated crRNA variant was not inferior to that of the canonical crRNA (Fig. 2d and Supplementary Fig. 7). The fluorescence intensity ratio normalized to the perfectly matched (PM) activator was significantly lower for FL1, FL2 and L1 compared to canonical crRNA, indicating that these three crRNA mutants exhibit improved specificity. This may have resulted from the target dsDNA binding defect of the RNP caused by a mutation in the DR sequence, as shown in Fig. 2a, which is a commonly used strategy to enhance the specificity of CRISPR-Cas effectors24,25.

Based on these results, the crRNA toolbox-guided Cas12a demonstrates diverse properties, offering researchers customizable tools that can be tailored to different scenarios, including gene editing and molecular diagnostics. Mutations in the DR sequence impact the binding affinity of the Cas12a-crRNA RNP to target dsDNA, as well as the cis- and trans-cleavage activities. This approach represents an effective strategy for regulating Cas12a functionality.

Tunable gene repression and precise base editing with crRNA toolbox

The results from the library screening have shown that mutating the DR sequence can reshape Cas12a-based CRISPRi into a tool for fine-tuning gene expression levels (Fig. 1). For the simplified crRNA toolbox, we conducted more detailed characterizations to assess its potential for improving the tunability of CRISPRi. The expression of crRNA and dCas12a was driven by J23119 and the arabinose-inducible araBAD promoter (PBAD), respectively. The RNP targeted gfp in the genome of Escherichia coli NEB 10-beta to suppress its expression (Fig. 3a). We employed two ribosome binding sites (RBS) to regulate dCas12a expression: the stronger RBS0 and the weaker RBS3326. Whether employing the RBS0 or RBS33 upstream of the dCas12a coding region, canonical crRNA-guided dCas12a exhibited leaky suppression in the absence of the inducer (Fig. 3b and Supplementary Figs. 8, 9). This issue of leaky repression, caused by promoter leakage, is a common challenge for CRISPRi systems, regardless of whether the Cas effector or gRNA is expressed under inducible promoters27,28,29. Notably, with the strong RBS, dCas12a guided by L1, FL1, and FL2 was less affected by leaky expression compared to dCas12a guided by canonical crRNA. In the absence of an inducer, the relative GFP expression level of these three crRNA remained at approximately 80% (Fig. 3b and Supplementary Fig. 8). Furthermore, when using the weak RBS, no leaky repression was observed with L1 and FL1 (Fig. 3b and Supplementary Fig. 9). Overall, the crRNA toolbox mitigates the leaky repression in CRISPRi without the need for complex genetic circuits, making it a more manageable tool for expression regulation.

Fig. 3: Tunable gene repression and precise base editing with crRNA toolbox.
figure 3

a Genetic components for the validation of tunable CRISPRi facilitated by crRNA toolbox. DR-mutated crRNAs guided dCas12a to inhibit gfp transcription in diverse levels, indicating tunable expression repression. The expression of dCas12a was controlled by a PBAD promoter. b Relative expression derived from seven crRNAs with no inducer (L-ara) added. A strong RBS (RBS0) and a weak RBS (RBS33) were used upstream of the dCas12a coding region. c Relative expression derived from seven crRNAs with various concentrations of inducer (L-ara: 0, 2, 5, 10 mM) added. d Relationship between gfp relative expression levels when crRNA mutants target different protospacers. Relative expression was calculated based on specific fluorescence levels (RFU/OD600) and normalized with the non-targeted control set to 100%. CN indicates canonical crRNA. e Genetic components for precise base editing facilitated by crRNA toolbox. DR-mutated crRNAs guided APOBEC-dCas12a-UGI/evoCDA-dCas12a-UGI for base editing and achieved tunable editing window. BE indicates base editor. f,g C to T editing efficiency at multiple sites derived from APOBEC-dCas12a-UGI (f) and evoCDA-dCas12a-UGI (g) guided by crRNA mutants. h,i Average C to T editing efficiency across the 20-bp spacer derived from APOBEC-dCas12a-UGI (h) and evoCDA-dCas12a-UGI (i) guided by crRNA mutants. Editing window narrowed with the use of FL2 and L1, indicating precise editing. Data are shown as mean ± s.d. for n  =  3 biologically independent samples. Source data are provided as a Source Data file.

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With the addition of L-arabinose (L-ara) at varying concentrations (0, 2, 5 and 10 mM), dCas12a guided by L1 exhibits a broader dynamic range of expression regulation compared to canonical crRNA (Fig. 3c and Supplementary Fig. 9). The relative expression level of the target gene responds more sensitively to changes in inducer concentration. Moreover, at the same L-ara concentration, the crRNA toolbox enabled varying levels of expression repression, with relative gfp expression ranging from 98.59 to 48.1% (Fig. 3c and Supplementary Fig. 9). When targeting different sites, the expression repression induced by the same crRNA mutant exhibited a consistent correlation (Fig. 3d and Supplementary Fig. 9, 10. The X-axis in Fig. 3d corresponds to spacer 1 used in Fig. 3b, c, while the Y-axis represents spacer 2.). This further demonstrates that DR mutations facilitate fine-tuning of repression levels in CRISPRi, acting as a sensitive knob rather than a simple on/off switch. As a representative collection of DR mutants, the crRNA toolbox provides researchers with a versatile and user-friendly set of options for precise expression control.

Similar to CRISPRi, the Cas12a base editor depends on a DNase-dead RNP to bind target dsDNA. Higher affinity for target dsDNA results in a wider editing window, enabling more effective termination of gene expression and the construction of mutation libraries30. In contrast, weaker binding to target dsDNA leads to a narrower editing window, allowing for precise introduction of mutations at specific locations. Currently, precise base editing tools tailored for prokaryotes remain limited, and those developed for eukaryotic systems may not be readily applicable to bacterial cells. For example, hA3Aa—a cytidine deaminase designed for high-precision editing—exhibited poor editing efficiency at all five tested genomic loci in the E. coli NEB 10-beta strain (Supplementary Fig. 11)31. This highlights the need for effective strategies to narrow the editing window, particularly in prokaryotic contexts. Differences in dsDNA binding ability among crRNA mutants may present alternative opportunities for regulating the editing window. Thus, we explored using the crRNA toolbox to guide the Cas12a cytidine base editor (CBE). We fused rat-derived cytidine deaminase APOBEC or the evolved version of lamprey CDA (evoCDA) to the N-terminal of dCas12a and UGI to the C-terminal, and guided the CBEs with 7 members of the crRNA toolbox (Fig. 3e)32. In a series of edits targeting different sites in the Escherichia coli NEB 10-beta genome, CBEs guided by different crRNAs exhibited distinct editing windows and C-to-T efficiency (Fig. 3f, g and Supplementary Fig. 12, 13). For APOBEC-dCas12a-UGI, the width of the editing window positively correlated with C-to-T conversion efficiency, likely due to changes in target dsDNA affinity caused by crRNA mutations (Fig. 3f). Specifically, lower-affinity crRNAs (e.g., L1, FL2) tended to produce narrower windows, whereas higher-affinity variants (e.g., canonical, F1) supported broader ones (Fig. 3f). This finding aligns with previous studies, which confirmed that stronger binding affinity of a CBE to its substrate leads to higher editing efficiency and a broader editing window30. Thus, for L1 and FL2, although their editing windows narrowed from positions 8-13 (for canonical crRNA) to positions 10-12, allowing for more precise editing, this came at the expense of reduced editing efficiency (Fig. 3h). In contrast to the peaking editing efficiency of 66% achieved with the canonical crRNA, L1 and FL2 exhibited peaking editing efficiencies of only around 30%.

Nevertheless, when paired with the more efficient deaminase evoCDA, crRNA mutants that exhibited suboptimal performance with APOBEC showed improved editing efficiency (Fig. 3g and Supplementary Fig. 13). This suggests that using high-efficiency deaminases allows the crRNA toolbox to achieve effective editing while maintaining control over the editing window. At specific sites, the crRNA mutants achieved editing efficiencies that exceeded those of the canonical crRNA, indicating that the crRNA toolbox offers more options for high editing efficiency (Fig. 3g). Furthermore, the editing window of L1 and FL2 was narrowed from five nucleotides (positions 8–11 and 16 for canonical crRNA) to three nucleotides (positions 8–10) (Fig. 3i). For FL2, this narrowing of the editing window allowed for precise editing without compromising efficiency, achieving levels comparable to the canonical crRNA.

When comparing APOBEC-dCas12a-UGI with evoCDA-dCas12a-UGI, changes in deaminase resulted in a shift in editing efficiency (Fig. 3f–i). The crRNA toolbox could be paired with various cytosine deaminases to target narrowed editing windows at different positions within the protospacer. Collectively, these results indicate that DR mutations can modulate the editing window of Cas12a base editors, offering additional opportunities for efficient editing across multiple sites and providing a distinct strategy for precise base editing.

Nevertheless, the crRNA toolbox provides a tool for precise base editing but does not offer substantial improvement in editing efficiency compared to the canonical crRNA. At certain sites (site 3 in particular), all crRNA variants exhibited relatively low editing efficiencies (Supplementary Fig. 12, 13). We hypothesized that variants with stronger GFP repression in the initial library screen might have enhanced DNA-binding capabilities, potentially improving editing outcomes. To test this, we selected two highly repressive variants, D20 and D21 (referred to as L3 and L4), and used them to guide APOBEC-dCas12a-UGI and evoCDA-dCas12a-UGI for base editing at previously inefficient sites (Fig. 1c and Supplementary Fig. 2). L3 and L4 significantly boosted editing performance—by up to 2.46-fold at low-efficiency sites (i.e., site 1 and site 5; Supplementary Fig. 14), and achieving editing rates of up to 57.7% at sites uneditable by canonical crRNA (i.e., site 3; Supplementary Fig. 14). These findings further underscore the flexibility and versatility of the crRNA mutation strategy: as the editing goal shifts from precision to efficiency, optimal variants—possibly beyond those included in the original toolbox—can be readily selected from the mutation library to meet evolving application needs.

Enhanced genome editing through crRNA-toolbox-mediated homologous recombination

CRISPR-mediated homologous recombination is a widely used strategy for genome editing in prokaryotes. The CRISPR system could induce double-strand breaks (DSBs) in the chromosome through its cis-cleavage activity. These breaks are then repaired via homologous recombination using an exogenous repair template (RT), enabling flexible knockouts, knock-ins, and replacements. However, DSBs can be lethal to bacteria unless repaired promptly. This lethal effect of Cas effectors results in a limited number of transformants and reduced editing efficiency33. Therefore, achieving gene knockouts and knock-ins in prokaryotes through CRISPR-mediated homologous recombination remains in need of further optimization. A recent study has shown that guiding SpCas9 with separate tracrRNA and crRNA, rather than a single sgRNA, can mitigate the lethal effect caused by excessively cis-cleavage, significantly enhancing transformation and editing efficiency12. Following this concept, we sought to improve editing efficiency by modulating intracellular Cas12a activity through alternative means. Specifically, we tested two approaches: (1) reducing Cas12a expression by lowering the concentration of the inducer, and (2) designing crRNAs to target suboptimal PAM sites (e.g., CTTA, TCCA), which are known to support lower Cas12a activity8. These methods were intended to attenuate Cas12a-mediated cleavage and thus potentially promote more efficient homologous recombination. However, neither approach resulted in noticeable improvements in transformation efficiency or genome editing outcomes (Supplementary Fig. 15), suggesting that such strategies may lack the precision or effectiveness required to fine-tune Cas12a activity for prokaryotic genome editing. In contrast, our crRNA variant toolbox allows for more controlled modulation of Cas12a activity across a broad dynamic range, providing a more effective means of optimizing gene editing conditions. We therefore propose that selecting appropriate crRNA variants from the toolbox represents an effective strategy for optimizing CRISPR-mediated homologous recombination.

To validate this hypothesis, we introduced the p15A plasmid, containing both the crRNA and RT, into E. coli NEB 10-beta carrying a Cas12a plasmid through a standard plasmid transformation assay. The target sequence was within gfp, where we aimed to knock out a 500 bp fragment or knock in a 1200 bp mcherry gene under the control of the Plac promoter (Fig. 4a). We assumed that crRNAs with stronger cis-cleavage activity would lead to increased cell death, resulting in fewer transformants, whereas crRNAs with weaker cis-cleavage activity would yield more transformants. Indeed, for most crRNAs except for FL1, the number of transformants was inversely correlated with their cis-cleavage efficiency (Fig. 4b, c and Supplementary Figs. 16, 17). When cis-cleavage efficiency fell below a certain threshold, the number of transformants surged by two orders of magnitude (L1, FL2), reaching or even surpassing levels seen with non-targeted controls (Fig. 4b, c and Supplementary Fig. 16). Reducing the targeting strength of Cas effectors likely buys time for homologous recombination, thereby increasing the likelihood that bacteria survive the DSB. This finding aligns with previous reports12,13. However, FL1 was an exception within the crRNA toolbox. Despite showing low cis-cleavage activity in vitro, FL1 yielded few transformants (Fig. 4b, c and Supplementary Figs. 16, 17). This may be attributed to the dual cis- and trans-cleavage activities of Cas12a, which complicate its mechanisms compared to Cas9. While cis-cleavage activity is a key factor influencing transformation efficiency, it may not be the sole determinant. Furthermore, unlike other crRNAs, transformants generated using L1 and FL2 exhibited varying colony sizes (Supplementary Fig. 16). We found that the larger colonies had not undergone the intended edits, suggesting they might have evaded Cas12a cleavage, allowing them to maintain faster growth.

Fig. 4: Activity-attenuated crRNA enhances transformation and editing efficiency for CRISPR-mediated homologous recombination.
figure 4

a Schematic of the experimental setup for CRIPSR-mediated homologous recombination aimed at fragment knockouts and knock-ins. RT, repair template. b,c Genome editing assay in E. coli NEB 10-beta-gfp to knock out 500 bp within gfp (b) or knock in 1200 bp mcherry-expressing fragment (c). Each dot for the transformations represents a single biological replicate. NT indicates non-targeting gRNAs. Each dot for the editing efficiencies represents the average of 16 colonies screened from one biological replicate. Data are shown as mean ± s.d. for n  =  3 biologically independent samples. d Phenotypic characterization of knockout and knock-in strains. Confirmed knockout and knock-in strains were cultured, resuspended in PBS buffer, and imaged using fluorescence microscopy. The left panel presents a schematic showing the effects of knockout and knock-in, while the right panel shows fluorescence microscopy images, confirming that the phenotypes of the knockout and knock-in strains were as expected. Scale bar: 10  μm. Representative micrographs of wild type (WT) strains were derived from a single experiment without replication. KO indicates knockout. KIN indicates knock-in. Source data are provided as a Source Data file.

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Next, we conducted colony PCR using primers flanking the genomic target and annealing outside the homology arms to assess editing efficiency (Supplementary Fig. 18, 19). Amplicon sequencing confirmed that fragment lengths corresponded to successful knockouts and knock-ins, and the phenotypes of the edited strains matched expectations (Fig. 4d and Supplementary Figs. 18, 19). Canonical crRNA was ineffective for achieving the 500 bp knockout and performed poorly in the 1200 bp knock-in (Fig. 4b, c). Among the six crRNA mutants in the toolbox, L1 showed superior performance in both knockout and knock-in experiments (Fig. 4b, c). The efficiency of the 500 bp knockout and 1200 bp knock-in using L1 reached 58.3 and 52.1%, respectively, which is 13.8 and 2.5 times higher than that of the canonical crRNA (Fig. 4b, c). This further supports that attenuating the cis-cleavage activity of Cas effectors not only increases transformant yields but also enhances editing efficiency. Given the substantial difference in the number of transformants, using L1 can produce 300 to 900 times more edited colonies in a single transformation experiment compared to the canonical crRNA. As researchers have noted, balancing CRISPR-Cas activity with host homologous recombination is crucial for effective genome editing in prokaryotic cells12. Our DR mutation strategy offers a controllable approach to regulate CRISPR-Cas activity, with the success of the crRNA toolbox in E.coli NEB 10-beta serving as a proof-of-concept.

Semiquantitative one-pot molecular diagnostics facilitated by crRNA toolbox

CRISPR-based molecular diagnostics typically rely on Cas proteins to recognize and cleave target nucleic acid, followed by trans-cleavage of nucleic acid probes for signal generation (Fig. 5a)4,34,35. To ensure high sensitivity, CRISPR diagnostic (CRISPR-Dx) is often paired with nucleic acid amplification (Fig. 5a)4,14,15,34,35,36. However, integrating amplification and CRISPR-Dx into a one-pot reaction—essential for simplifying workflows and preventing aerosol contamination—poses a challenge. Cas effectors can prematurely cleave the target nucleic acids as amplification and cis-cleavage compete for the substrate, leading to weak signal output and a high limit of detection (LoD) (Fig. 5b)37,38. One strategy to lower the LoD in one-pot detection is to reduce the cis-cleavage activity of Cas effectors, thereby balancing amplification and cleavage14. This can be achieved through recognition of suboptimal PAMs, as shown in a previous studies15. However, reliance on suboptimal PAMs limits target site selection and presents a drawback for one-pot detection at canonical PAM sites. Also, selecting for an appropriate suboptimal PAM can complicate the development of detection methods39. Another strategy to lower the LoD in one-pot detection involves caged crRNAs paired with a protective oligo, which activate cleavage only upon light exposure40. These chemical modifications and light activation modules, however, increase detection costs and complicate the system. Additionally, one-pot detection faces challenges in generating quantitative outputs. To address these issues, we took Mycoplasma pneumoniae as a model and utilized the crRNA toolbox to develop an ultra-simple semiquantitative one-pot detection method.

Fig. 5: crRNA toolbox-based semiquantitative one-pot Cas12a detection method.
figure 5

a Schematic of one-pot Cas12a nucleic acid detection. b Schematic of regulating Cas12a cis-cleavage activity with crRNA toolbox to modulate detection sensitivity. c–f, Characterization of the LoD of the one-pot RPA-CRISPR-Dx using canonical crRNA (c), L1 (d), FL2 (e), and F1 (f) targeting the P1 adhesin gene of M. pneumoniae. Fluorescence at 30 min of the detection assay with background subtracted was used for LoD assessment. The gray dashed lines indicate the threshold, set as the negative control plus 3 times its standard deviation. g–i Specific characterization of crRNA toolbox-assisted one-pot detection. Detection systems containing L1 (g), FL2 (h), and F1 (i) only showed positive results for M. pneumoniae-derived nucleic acids, confirming their specificity. j Schematic of the semiquantitative strategy using crRNA toolbox. One-pot detection systems with different LoDs are included in an array. For a given sample, three detection systems in the array provide independent results and the number of positive results indicates the concentration range. k,l Qualitative results using the most sensitive detection system containing F1 for 40 M. pneumoniae- positive sputum or aspirates samples (k) and 15 M. pneumoniae-negative samples (l). The gray dashed lines denote the threshold. qPCR serves as the gold standard for determining negative and positive results. m Sensitivity (true positive rate) defined as: Sensitivity = True Positives/(True Positives + False Negatives). Specificity (true negative rate) defined as: Specificity = True Negatives/(True Negatives + False Positives). n–p Detection of 10 M. pneumoniae-positive sputum or aspirate samples using one-pot detection systems containing L1 (n), FL2 (o), and F1 (p). The gray dashed lines represent the threshold, with qPCR used as the gold standard for determining target nucleic acid concentration. Semiquantitative results are derived by comparing the results from the three detection systems for the same sample. q Comparison between qPCR results and SONAR semiquantitative results. SONAR-determined target nucleic acid concentrations are consistent with qPCR results. Data are shown as mean ± s.d. for n  =  3 biologically independent samples. Statistical significance was analyzed using a two-tailed t test: ns, p  >  0.05. Source data are provided as a Source Data file.

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M. pneumoniae is a common cause of respiratory infections, leading to periodic outbreaks every few years41,42. Following the decline of the COVID-19 pandemic, a global surge in M. pneumoniae cases has emerged43. To mitigate the impact of this resurgence and prepare for future outbreaks, it is crucial to develop a rapid point-of-care testing (POCT) method. We selected the P1 adhesin gene, a commonly used target for nucleic acid detection of M. pneumoniae, and utilized the nine crRNA mutants (L1, L2, FL1, FL2, F1, F2, along with the high-activity mutants L3 and L4) to guide Cas12a for target recognition. The CRISPR-Dx was coupled with recombinase polymerase amplification (RPA) to ensure sensitivity. Consistent with previous findings, the use of canonical crRNA in one-pot RPA-CRISPR-Dx resulted in a relatively high LoD (Fig. 5c and Supplementary Fig. 20a)15. For similar reasons, the high-activity crRNA mutants L3 and L4 did not confer advantage in one-pot RPA-CRISPR-Dx, with LoD comparable to that of the canonical crRNA (Supplementary Fig. 20b, c). In contrast, the application of relatively low-activity crRNA mutants significantly improved sensitivity, delivering rapid results within 30 min, with each mutant yielding distinct LoDs (Fig. 5d-f and Supplementary Fig. 20). Specifically, the LoDs for L1, FL2 and F1 were 100 aM, 10 aM, and 1 aM, respectively (Fig. 5d-f). Notably, F1, which achieved the lowest LoD, demonstrated a sensitivity comparable to that of quantitative PCR (qPCR). Interestingly, the detection sensitivity did not directly correlate with the cis- or trans-cleavage activities of the crRNA mutants. Although mutated crRNAs with lower cis-cleavage activity generally showed higher sensitivity compared to the canonical crRNA, lower cis-cleavage activity did not always translate to higher sensitivity. This suggests that in one-pot CRISPR-Dx, optimizing the balance between amplification and cis-cleavage is critical for enhancing the sensitivity (Fig. 5b). Meanwhile, multiple factors, despite cis-cleavage might contribute to the overall performance of Cas12a in nucleic acid detection.

Next, we tested the specificity of the three detection systems containing L1, FL2 and F1 with nucleic acids from seven pathogenic microbes—monkeypox virus (mpox), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), E. coli, Pseudomonas aeruginosa, Pseudomonas putida, Shewanella putrefaciens, and Helicobacter pylori. All assays returned negative results, confirming that CRISPR-Dx system using these crRNA mutants maintained high specificity (Fig. 5g–i).

To achieve semiquantitative detection, we designed an array comprising the three detection systems with different LoDs (L1: 100 aM, FL2: 10 aM, F1: 1 aM). These systems are applied simultaneously to the same sample, with the number of positive results corresponding to the target nucleic acid concentration. A single positive result indicated a concentration between 1–10 aM, two positives signaled 10–100 aM, and all three positives indicated a concentration exceeding 100 aM (Fig. 5j). We named this approach “Semiquantitative One-pot CRISPR-Dx based on Noncanonical crRNAs” (SONAR).

We collected 55 clinical samples of sputum or lung aspirates and tested them using both the validated qPCR method and our SONAR approach44. The qPCR results indicated that 40 of the 55 samples were positive for M. pneumoniae, while 15 were negative (Supplementary Fig. 21). Using the most sensitive detection system containing F1, we performed qualitative testing on all samples. Of the 40 positive samples, 39 were reported as positive by the F1 system, one positive sample was incorrectly reported as negative, and all 15 negative samples were confirmed as negative. This yielded a sensitivity of 97.5% and a specificity of 100% (Fig. 5k-m). We then conducted semiquantitative testing on 10 of the positive samples using the detection array, with results consistent with the qPCR findings (Fig. 5n–q and Supplementary Fig. 21, 22). Of the samples, three had target concentrations of 1–10 aM, three ranged from 10 to 100 aM, and four exceeded 100 aM. These results indicate that SONAR enables rapid and accurate semiquantitative detection of M. pneumoniae.

The unique advantage of the crRNA toolbox lies in its diversity, enabling fine-tuned control over the balance between amplification and cleavage to adjust sensitivity (Fig. 5b). This allows for an exceptionally simple one-pot detection strategy with sensitivity comparable to qPCR without additional modifications, substances, or changes to the target site. The RPA-CRISPR-Dx is inherently a POCT solution that does not require specialized instrumentation, and SONAR takes this simplicity a step further by enabling rapid semiquantitative result reading without the need for processing or calculations. Among existing technologies, SONAR excels in detection time and LoD while offering the distinctive advantage of semi-quantitative results (Table 2). Our approach holds promise for home diagnostics, bedside testing, and use in resource-limited settings.

Table 2 Comparison among Cas12-based one-pot detection methods
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Discussion

Within the rapidly progressing CRISPR field, there is a growing demand for more precise control over Cas protein activity. As a tool for nucleic acid targeting and cleavage, CRISPR system often operates in conjunction with other processes, such as nucleic acid amplification in diagnostics or homologous recombination in gene editing, while also adapting to diverse in vivo and in vitro conditions12,15,45. These complex scenarios call for a more versatile and customizable set of CRISPR tools. In this study, we demonstrate that crRNA DR sequence mutations can serve as a flexible strategy for regulating Cas12a activity. Given the large size and structural complexity of Cas12a, the ability to modulate its activity through just a few nucleotide substitutions in crRNA highlights an elegant and efficient regulatory mechanism. DR sequence mutants are not only easy to generate but also highly diverse, providing expanded options for fine-tuning Cas12a function. Compared to existing Cas12a crRNA engineering strategies, our method offers a uniquely flexible, cost-effective, and straightforward means of modulating Cas12a activity, broadening its applicability across various contexts (Table 1).

Prior research primarily designed targeted mutants to enhance Cas12a performance in specific applications, such as inducing indels or improving nucleic acid detection8,10,16,17,46. In this study, we curated a set of crRNA mutants with distinct properties to establish a crRNA toolbox. By harnessing the complementarity and synergy among the mutants, we effectively optimized Cas12a performance across diverse application scenarios (Fig. 6). As an expression regulation tool, the crRNA toolbox allows CRISPRi to bypass issues of leaky repression, providing more precise and fine-tunable gene suppression. In base editing, the crRNA toolbox-assisted Cas12a CBE enables precise edits. Its compatibility with various deaminases allows for targeted modifications across different regions within the spacer. For CRISPR-mediated homologous recombination, the toolbox offers options to balance host homologous recombination with CRISPR cleavage, improving transformation and editing efficiency. In nucleic acid diagnostics, the toolbox introduces a semiquantitative one-pot strategy by regulating the balance between nucleic acid amplification and CRISPR-Dx, delivering rapid and accurate semiquantitative results ideal for POCT applications. Furthermore, when addressing a broader range of application demands (e.g., base editing with enhanced efficiency in Supplementary Fig. 14), our approach enables the efficient selection of the optimal crRNA mutants with minimal trial-and-error effort. Overall, DR sequence mutations and the resulting crRNA toolbox offer not only a distinct conceptual approach but also a practical, efficient, and highly accessible solution.

Fig. 6: The DR-mutated crRNA toolbox provides an adjustable, controllable, and flexible approach to regulating Cas12a activity.
figure 6

Each crRNA mutant generated through DR mutations offers unique advantages, making this toolbox highly adaptable for diverse applications, including gene expression regulation, base editing, fragment editing, and nucleic acid detection. The toolbox upgrades Cas12a’s capabilities by enabling fine-tuned expression repression, precise base editing, efficient fragment knock-in/knockout, and semi-quantitative one-pot detection.

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However, we have also observed that the performance of crRNA mutants in a simplified system does not always correlate directly with their applicability to specific contexts (Fig. 2). For instance, while most crRNA mutants with reduced cis-cleavage activity alleviate Cas12a toxicity in prokaryotes, thereby improving cell survival after DSBs in the genome, FL1 stands out as an exception. In the realm of molecular diagnostics, crRNA mutants that offer the highest sensitivity are not necessarily those with the lowest cis-cleavage activity. The functional complexity of Cas12a—encompassing its crRNA binding, processing, and both cis- and trans-cleavage of substrates3,4,6—necessitates further investigation to establish qualitative and quantitative relationships among crRNA sequences, Cas12a activity, and applicability in various scenarios. Computational modeling can aid in elucidating these intricate mechanisms. Given the current uncertainties surrounding this process, machine learning may offer insights to illuminate this black box. Looking ahead, we aspire to shift from the paradigm of screening crRNA mutants to designing DR sequences, thereby expanding our options for controlling and enhancing Cas12a.

The user-friendly, adjustable, controllable, and flexible crRNA toolbox has the potential to inspire further advancements in CRISPR technology. Previous studies have explored combining crRNA modifications with engineered Cas12a proteins, showing promising synergies11,47. The crRNA toolbox is not intended to replace or negate protein engineering but to complement it. Future directions may involve integrating the toolbox with engineered Cas12a, deaminases, and other regulatory proteins. Furthermore, our study has only characterized and applied a couple of representative crRNA mutants, yet the DR mutation library contains numerous other promising candidates. These unexplored mutants represent a valuable reservoir for future research endeavors. It is also worth noting that, while the present study mainly investigates bacterial and in vitro applications of the crRNA toolbox, the same strategy is anticipated to be equally valuable as a versatile tool for dynamic modulation of Cas12a activity in eukaryotic systems. Taken together, the crRNA toolbox, characterized by low implementation and optimization costs as well as broad adaptability, offers considerable potential for expansion across diverse biological contexts.

The DR mutation strategy has already demonstrated its strong capability to regulate Cas effectors. Some studies have shown that mutations in the gRNA handle can modulate the performance of dCas9 and dCas13 in CRISPRi48,49. We hypothesize that this strategy could be extended to other Cas effectors, possibly even to class 1 CRISPR systems. However, the fidelity requirements for gRNA handle regions may vary between Cas effectors, so the generalizability of this strategy will require further experimental validation50.

In conclusion, the DR mutation strategy has expanded the CRISPR toolbox and is likely to continue doing so. Its key advantages—adjustability, controllability, flexibility, and ease of use—make it a powerful complement to existing Cas effector regulation methods.

Methods

Ethical statement

All experiments involving saliva and sputum samples were approved by the Ethics Committee of the Fujian Medical University Union Hospital, China (2024KY093). All patients provided informed written consent. No participant compensation was provided.

Bacterial strains, plasmids, and reagents

Supplementary Information contains all strains, plasmids and oligonucleotides used in this work. The E. coli NEB 10-beta-gfp strain, characterized by a lac-promotor-driven gfp-lacZ expression cassette integrated in the genome, was produced through homologous recombination, following the methodology outlined in a previous study51. Specifically, the gene-editing plasmids encoding the homing endonuclease I-SceI and the λ-Red recombination system were introduced into target strains by conjugation or electroporation. Single-crossover colonies were identified by PCR and propagated in liquid Luria–Bertani medium. Genomic double-strand breaks and homologous recombination were subsequently induced, and the resulting cultures were plated. Final colonies were screened by PCR and confirmed by Sanger sequencing.

All plasmids were constructed using standard cloning techniques. After plasmid extraction, the concentration was measured using a Nanodrop spectrophotometer (Thermo Scientific). A total of 500 ng of plasmid DNA was used for each transformation across all experiments. The cultures for plasmid construction and strains were performed at 37 °C in 2 × yeast extract-tryptone (2 × YT) medium or on a 2 × YT agar plate containing corresponding antibiotics (50 μg/mL kanamycin, 34 μg/mL chloramphenicol, 100 μg/mL ampicillin) unless otherwise specified.

crRNA library construction

We commercially synthesized primers with randomized bases in the target mutation region of the crRNA DR sequence. To construct the plasmid library, fragments containing crRNA mutants were amplified using a template plasmid encoding the canonical crRNA that targeted gfp downstream of a J23119 promoter. The plasmid also contained dCas12a (D832A) under the control of a PBAD promoter, enabling knockdown of gfp. The pooled plasmid library was then transformed into E. coli NEB 10-beta-gfp to analyze repression efficiency.

CRISPRi repression efficiency

Plasmids containing dCas12a and the crRNAs were transformed into E. coli NEB 10-beta-gfp. After overnight incubation, the transformants were transferred into 2 × YT medium containing 10  mM L-ara and cultured in a microplate at 37 °C. Fluorescence intensity (excitation at 485 nm and emission at 520 nm) and cell density (OD600) were dynamically monitored using a BioTek Synergy HT Microplate Reader (BioTek Instruments Co., U.S.A.). Relative expression was calculated by normalizing fluorescence units to OD600, with the non-targeted control set at 100%. Relative expression at the 15 h time point was used to screen and compare repression efficiency.

LbCas12a protein expression and purification

The LbCas12a-encoding fragment was cloned into a pET-based expression vector containing a C-terminal 6 × His-tag. The E. coli strain Rosetta was transformed with the plasmid and incubated in Luria–Bertani (LB) medium. Once the OD600 reached 0.7, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and the culture was incubated at 16 °C for 16 h with shaking. The protein was then purified from the cell lysate using Ni-NTA resin and eluted with a buffer consisting of 50 mM Tris-HCl (pH 8.0), 1.5 M NaCl, 600 mM imidazole, and 5% glycerol. The purified protein was stored at −80 °C for future use. Protein concentration was measured using a modified Bradford protein assay kit (Sangon Bio, China).

Preparation of crRNAs

We synthesized two oligos as templates for in vitro transcription, incorporating the T7 promoter and the crRNA sequences. The oligos were heated to 95 °C for 4 min, then gradually cooled at a rate of 0.1 °C/s to 4 °C to form double-stranded DNA. The templates were incubated with T7 RNA polymerase (Vazyme Biotech Co., China) at 37 °C for 2 h to produce crRNA. DNase I (Vazyme Biotech Co., China) was subsequently added to digest the DNA template, with incubation at 37 °C for 15 min. The crRNA was then purified using an RNA purification kit (Jianshi Biotechnology Co., China). The crRNA sequence is provided in Supplementary Data 1.

Surface plasmon resonance binding analysis

SPR measurements were conducted at 25 °C using a Biacore 8000 instrument equipped with a CM5 sensor chip (GE Healthcare). Approximately 8000 response units (RUs) of LbCas12a were immobilized on the chip surface via standard amine coupling. Canonical crRNA and its mutant variants were prepared in running buffer and injected at concentrations ranging from 1.25 μM to 20 μM in a serial dilution series. Analytes were passed over the immobilized protein at a constant flow rate, and association/dissociation responses were recorded in real time. Following each binding cycle, the chip surface was regenerated with a brief injection of 10 mM NaOH to remove residual analyte. Kinetic parameters (association rate constant ka, dissociation rate constant kd, and equilibrium dissociation constant KD) were calculated using the BIAevaluation software.

Electrophoretic mobility shift assay

For investigating the interaction between the dCas12a-crRNA RNP and target dsDNA, dCas12a was expressed and purified as previously described. We synthesized 3’-FAM-labeled dsDNA substrates. The dCas12a, crRNA, and 5 nM dsDNA probe were mixed in Buffer-1 and incubated at 37 °C for 15 min. The mixture was then subjected to gel electrophoresis on a 5% polyacrylamide gel at 140 V for 25 min. Imaging was performed using the ImageQuant 800 system (Amersham Biosciences, UK). Sequences of oligonucleotides and probes used are listed in Supplementary Data 2.

In vitro cis- and trans-cleavage activity of Cas12a with various crRNA

For the characterization of cis-cleavage, 250 nM of pre-annealed dsDNA target labeled with FAM and BHQ1 was incubated with 50 nM LbCas12a and 200 nM crRNA in either Buffer-1 or rCutSmart Buffer (New England BioLabs) at 37 °C for 60 min. The fluorescence (excitation at 485 nm and emission at 520 nm) was dynamically monitored using the StepOne Real-Time PCR system (Applied Biosystems, Foster City, CA). Sequences of oligonucleotides and probes used are listed in Supplementary Data 2.

For the characterization of trans-cleavage, 100 pM, 1 nM or 10 nM pre-annealed unlabeled dsDNA substrate was incubated with 50 nM LbCas12a, 200 nM crRNA and an 8 C ssDNA probe labeled with FAM and BHQ1 in Buffer-1 at 37 °C for 60 min. The fluorescence was monitored as described above. Sequences of oligonucleotides and probes used are listed in Supplementary Data 2.

Base editing in E. coli NEB 10-beta

For base editing, we constructed a plasmid that expresses APOBEC-dCas12a-UGI/evoCDA-dCas12a-UGI under the control of a PBAD promoter, with crRNAs located downstream of a J23119 promoter. This vector was transformed into E. coli NEB 10-beta-gfp. After 24 h of incubation on an agar plate containing 10 mM l-ara, colonies were collected. We employed primers that anneal approximately 500 bp upstream and downstream of the target to amplify the genomic DNA of the strains. The resulting amplicons were sequenced via Sanger sequencing, and the C-to-T editing efficiency at the target site was calculated using the online tool Edit R52.

CRISPR-mediated homologous recombination

We constructed a p15A-based vector containing the J23119 promoter-driven expression of crRNA and an RT. The RT included two 1000 bp homology arms, designed to introduce either a 500 -bp knockout or a 1200-bp knock-in. This plasmid was transformed into E. coli NEB 10-beta-gfp, which harbored a Cas12a expression vector controlled by the PBAD promoter, via electroporation. After a 1 h recovery at 37 °C, the cells were plated in 10 -µL serial dilutions. After 20 h of incubation at 37 °C, the transformants were counted.

To determine the editing efficiency, we randomly selected single transformant colonies for PCR. Primers were designed to anneal outside the homology arms. The length of the PCR products was analyzed using 1% agarose gel electrophoresis to determine whether editing had occurred in each clone. The amplicons were then confirmed via Sanger sequencing. To verify the phenotypes of wild-type and edited strains, we selected three knockout and three knock-in clones, expanded the cultures, and resuspended the cells in PBS buffer. Imaging was performed using an inverted fluorescence microscope (Olympus, Tokyo, Japan).

qPCR

Commercially synthesized plasmids (General Bio Co., China) containing the P1 adhesin gene sequence of M. pneumoniae (Mycoplasmoides pneumoniae M129 accession: NZ_OU342337) were serially diluted and used as standard samples to generate a qPCR standard curve. The qPCR assay for M. pneumoniae detection was conducted in a 20 µL reaction mixture, consisting of 10 µL of 2 × AceQ qPCR Probe Master Mix (Vazyme Biotech Co., China), 1 µL of each primer (10 µM), and 0.2 µL of TaqMan probe (10 µM, Sangon Biotech Co., China). Details of the primers and TaqMan probes are listed in Supplementary Table 2.

Semiquantitative one-pot CRISPR-Dx based on noncanonical crRNAs

The plasmid containing the target sequence of M. pneumoniae, as described above, was used to determine the LoD. Lyophilized RPA particles (Weifang Amp-Future Biotech Co., China) were reconstituted in a reaction mixture consisting of 29.4 µL Buffer A, 2 µL each of 20 µM RPA forward and reverse primers, 400 nM 8 C ssDNA probe, 100 nM LbCas12a, 200 nM crRNA, and nuclease-free water. The 24 µL of this mixture was then combined with 3 µL Buffer B and 3 µL of the sample to be tested, followed by incubation at 37 °C. Fluorescence (excitation at 485 nm and emission at 520 nm) was monitored in real-time using the StepOne Real-Time PCR system (Applied Biosystems, Foster City, CA). Details of the primers and probes are listed in Supplementary Table 2.

Microbial Samples collection and processing

To validate the specificity of SONAR, interference samples including Mpox pseudovirus (commercially synthesized), SARS-CoV-2 (Fujian Medical University Union Hospital, China), E. coli (Laboratory storage), Pseudomonas aeruginosa (Laboratory storage), Pseudomonas putida (Laboratory storage), Shewanella putrefaciens (Laboratory storage), and Helicobacter pylori (Guangdong Microbial Culture Collection Center, China) were collected. M. pneumoniae samples, including sputum and lung aspirates from 55 patients, were collected at the Fujian Medical University Union Hospital, China. The samples used in this study were de-identified and no demographic information, including sex and/or gender, was collected. Accordingly, sex and/or gender were not considered in the study design. Samples were stored at 0–4 °C and transported to the laboratory for analysis within 48 h. All samples were inactivated at 65 °C for 2 h, after which DNA was extracted and purified using a DNA extraction kit. The resulting products were stored at −80 °C for future analysis.

Statistics & reproducibility

Statistical analyses were carried out using Microsoft Excel (2016) and Origin Pro (v9.5.0 SR1). Differences between groups were assessed with a two-tailed t-test, applying a significance threshold of p < 0.05. Random sampling was used for all M. pneumoniae, SARS-CoV-2, E. coli, Pseudomonas aeruginosa, Pseudomonas putida, Shewanella putrefaciens, and Helicobacter pylori samples. Sample size was not predetermined by statistical methods. No data points were excluded from analysis. Experiments were not randomized, and investigators were aware of group allocation during experimentation and outcome evaluation.

Reporting summary

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