Based on our reading, no studies have yet demonstrated the capacity of CRISPR-Cas13a to directly detect DNA. Researchers commonly use T7 transcription to convert DNA into RNA, thereby enabling the CRISPR-Cas13a system to detect DNA through RNA intermediates12, 22, 30. However, through trans-cleavage reporter assays, we discovered that Cas13a crRNA can effectively recognize and bind to both ssDNA and double-stranded DNA (dsDNA), thereby initiating the trans-RNase activity of Cas13a, which leads to the collateral cleavage of fluorophore/quencher-labelled RNA reporters (FAM-rUrUrUrUrUrUrUrUrU-BHQ1) (Fig. 1). The minimum detectable DNA concentration was 0.1 nM. Notably, ssDNA demonstrated a more rapid and pronounced increase in fluorescence intensity upon activation compared to dsDNA.
The Cas13a crRNA typically includes a spacer region that matches the target RNA sequence, and a direct repeat sequence with a stem-loop structure necessary for crRNA stability and Cas13a interaction31. Typical spacer lengths of Cas13a crRNAs for gene knockdown and nucleic acid detection range from 20 to 28 nucleotides (nt)10, 12, 31. To evaluate the effect of spacer length on Cas13a’s ability to detect DNA targets, we synthesized crRNAs with varying spacer lengths (17, 20, 23, and 28 nt) (Fig. 2a). Cas13a showed significant activation targeting RNA at various concentrations (100 nM, 10 nM, 1 nM) with these crRNAs (Fig. 2b). In contrast, spacer length markedly influenced its trans-RNase activity towards DNA targets, with shorter lengths diminishing the enzyme’s activity. Importantly, crRNAs with a 17 nt spacer did not trigger Cas13a’s trans-RNase activity (Fig. 2c).
We further investigated the impact of DNA targets on the sensitivity of Cas13a’s trans-RNase activity to single-base mismatches. Recent studies have identified position 7 (+ 7) within the crRNA spacer:RNA target duplexes as the most sensitive mismatch hotspot for Cas13a32. To ascertain the effects of varying target concentrations (10 nM, 1 nM) and crRNA spacer lengths (23, 28 nt) on Cas13a’s trans-RNase activity, identical sequences of RNA and ssDNA targets, each with a single-base mismatch at position 7 (+ 7), were synthesized (Fig. 2a). This assessment involved measuring the fluorescence intensity ratio of single-base mismatch (MM) targets to perfectly matched (PM) targets (Fig. 2d). We discovered that a single-base mismatch within RNA targets minimally impacts Cas13a’s trans-RNase activity, as evidenced by the nearly equivalent ratio of endpoint fluorescence intensities (time = 120 min), ranging from 0.85 to 0.97. In contrast, Cas13a exhibited pronounced sensitivity to single-base mismatches in ssDNA targets (ssDNA-MM), especially when paired with crRNAs of a 23 nt spacer length. This setup led to a reduction in Cas13a’s trans-RNase activity by over 79% compared to perfectly matched ssDNA (ssDNA-PM) targets. These findings demonstrate that employing ssDNA as detection targets significantly enhances CRISPR-Cas13a’s sensitivity to single-base mismatches.
Intrinsic RNase activity of Cas12f1 independent of activation
The CRISPR-Cas12a system demonstrates non-specific cleavage of both DNA and RNA (trans-DNase and trans-RNase activities) upon activation by crRNA:DNA target and crRNA:RNA target duplexes6, 7, 18, 19, 20, 21 (Supplementary Fig. 1). Employing RNA reporters with various sequences (rCrCrCrCrCrC, rArArArArArA, rUrUrUrUrUrU, rArArUrUrUrA, rUrUrUrUrUrUrUrUrU), we noted that the trans-RNase activity of Cas12a efficiently cleaved a wide range of RNA reporter sequences, notably including polyU sequences, which are commonly targeted by CRISPR-Cas13a’s trans-RNase activity (Supplementary Fig. 2b, c). Motivated by these findings in Cas12a, we sought to determine whether Cas12f1 exhibits a similar DNA and RNA target-activated trans-RNase and trans-DNase activity.
Cas12a and Cas13a gRNAs consist solely of short crRNA sequences11, 33. In contrast, Cas12f1’s gRNA comprises not only crRNA but also an elongated trans-activating crRNA (tracrRNA), engineered together into a single-guide RNA (sgRNA)9. The standard sgRNA length for Un1Cas12f1 is 220 nt9, which exceeds the capabilities of traditional chemical synthesis. As a result, we adopted a streamlined sequence featuring a 20 nt target-complementary region and a 100 nt scaffold34. Furthermore, Cas12f1 has been identified as the most compact Class 2 CRISPR-Cas effector to date, with sizes ranging from 400 to 700 amino acids9. Cas12f1’s functionality necessitates sgRNA-mediated dimerization14, 15. The requirement for dimerization is thought to compensate for its relatively small size.
It is widely recognized that Cas12f1 exhibits DNA and RNA-target activated trans-DNase activity9, 35, 36. In our trans-cleavage reporter assays, we found that Cas12f1 displays intrinsic RNase activity, independent of activation by sgRNA:ssDNA duplex activators (Fig. 3a, b). This finding contrasts with Cas12a, which necessitates duplex activators for its trans-RNase activity (Supplementary Fig. 1d, e). Furthermore, our analysis indicated that Cas12f1 RNase more efficiently cleaves polyrC (rCrCrCrCrCrC) sequences compared to polyA (rArArArArArA), polyU (rUrUrUrUrUrU), and rArU (rArArUrUrUrA) sequences of identical length (Supplementary Fig. 2d, e). However, this efficiency is consistent with that observed for longer polyU sequences (rUrUrUrUrUrUrUrUrU), which are predominantly used in CRISPR-Cas13a assays in this study.
We further sought to identify key residues critical for the trans-RNase activity of Cas12a and Cas12f1. Cas12a possesses RNase activity, cleaving precursor CRISPR RNA (pre-crRNA) adjacent to a stem-loop structure in the direct repeats (DR), thus producing intermediate crRNAs that are further processed into mature crRNAs13. Moreover, the key residues of Cas12a involved in this process have been elucidated37. We hypothesized that the RNase domain might also facilitate target-activated non-specific RNA cleavage. To test this hypothesis, we aligned the amino acid sequences of 16 Cas12a orthologs and introduced mutations at three conserved residues within RNase domain (H759A, K768A, K785A) in LbCas12a (Supplementary Fig. 3). Mutations in each residue resulted in a significant reduction in trans-RNase activity (Supplementary Fig. 4b, c), without affecting the endpoint fluorescence intensity of trans-DNase activity after 120 minutes, despite a lower initial enzymatic rate (Supplementary Fig. 4d, e).
To elucidate key residues affecting Cas12f1’s RNase activity, we aligned the amino acid sequences of three proteins: Un1Cas12f1, FnCas12a, and LbCas12a (Supplementary Fig. 5), and performed point mutations at four amino acid sites (K173, K186, K196, K198). Additionally, we mutated D326, a residue previously verified to be crucial for Cas12f1’s DNase activity14, 15. The results indicated that mutations in each residue (K173A, K186A, K196A, K198A) substantially reduced Cas12f1’s intrinsic RNase activity. Conversely, mutating D326 in Cas12f1 enhanced its intrinsic RNase activity (Fig. 3c, d). Furthermore, these alterations (K173A, K186A, K196A, K198A) slowed the trans-DNase reaction rate in Cas12f1, with mutations at K173 and K198 notably diminishing the final intensity of Cas12f1’s trans-DNase activity after 120 minutes (Fig. 3e, f).
Non-conservative activation of Cas12a and Cas13a trans-cleavage activity
A number of studies have sought to integrate various CRISPR-Cas systems, targeting multiplexed detection22, 23, 24, 25, 26, 27, 28, 29 or eliminating the need for pre-amplification prior to detection38. A fundamental condition for integrating CRISPR-Cas systems into a single detection assay is avoiding cross-activation of Cas enzymes by gRNA:DNA/RNA target duplexes from differing systems. To investigating this, we assessed the cross-activation potential among CRISPR-LbuCas13a, LbCas12a, and Un1Cas12f1 systems using ssDNA as the detection target. We observed that the trans-cleavage activities of Cas13a and Cas12f1 were specifically activated by their respective systems’ gRNA:ssDNA duplexes, demonstrating significant specificity (Fig. 4a, b, g, h). In contrast, both the trans-DNase and trans-RNase activities of Cas12a were triggered by gRNA:ssDNA duplexes from all three systems, indicating a less activation specificity (Fig. 4c-f).
The stem-loop structures within the DR sequences of crRNAs play a pivotal role in the functional efficiency and fidelity of both Cas12a21, 39 and Cas13a40. The similarity in lengths and stem-loop structures of crRNAs across these systems suggests that Cas13a crRNA:ssDNA duplexes could initiate Cas12a’s trans-cleavage activity (Fig. 4c-f; Supplementary Fig. 6a, d). To explore this, we modified the DR sequences in Cas13a crRNA to remove the stem-loop structures and evaluated modified Cas13a crRNA’s (Cas13a crRNAmt) ability to activate Cas12a’s trans-DNase activity (Supplementary Fig. 6a). The results showed that removing stem-loop structures from Cas13a crRNA did not reduce its ability to activate Cas12a’s trans-DNase activity (Supplementary Fig. 6b, c). We hypothesize that heteroduplexes formed by the pairing of the spacer region (28 nt) of Cas13a crRNA or crRNAmt with the ssDNA target are essential for triggering Cas12a’s trans-DNase activity, suggesting that nucleic acid duplexes of diverse sequence could trigger this activity. To verify this hypothesis, we synthesized RNA random sequences (RS/RNA) of various lengths (30, 25, 20, 18 nt) without stem-loop structures, which were paired with fully complementary ssDNA and RNA sequences to form RS/RNA:DNA heteroduplexes and RS/RNA:RNA homoduplexes of different lengths (Fig. 5a). Trans-cleavage reporter analysis showed that RS/RNA-DNA heteroduplexes, 30 base pairs in length, effectively activated Cas12a’s trans-DNase activity. Conversely, RS/RNA-RNA homoduplexes, irrespective of their length, did not exhibit this capability (Fig. 5b). Similarly, mutations were introduced into the DR sequence of Cas12a crRNA to dismantle its stem-loop structure (Supplementary Fig. 6d). The mutated Cas12a crRNA (Cas12a crRNAmt), upon pairing with complementary RNA, effectively activated Cas13a’s trans-RNase activity (Supplementary Fig. 6e, f). Additionally, RS/RNA:RNA homoduplexes comprising 30, 25, and 20 base pairs significantly induced Cas13a’s trans-RNase activity. In contrast, RS/RNA:DNA heteroduplexes, regardless of their length, did not demonstrate this ability (Fig. 5c).
The presented data reveal that the trans-cleavage activities of Cas12a and Cas13a exhibit non-conservative activation, where Cas12a exhibits significant tolerance for RNA:DNA heteroduplex sequences, and Cas13a for RNA:RNA homoduplexes. However, despite the stem-loop structure of crRNA not being essential for activating their trans-cleavage functions, it is pivotal in determining the specificity towards the types of nucleic acids that can be detected. Specifically, while Cas12a and Cas13a are capable of utilizing RNA and DNA as detection targets, respectively, is contingent upon the presence of their respective crRNAs (Supplementary Fig. 7).
CRISPR-drCas12f1/Cas13a-based dual-gene detection of IVA/H1N1 virus and human POP7 gene
The results presented above indicate that: (1) The CRISPR-Cas13a system can directly target DNA, thereby eliminating the need for additional reagents or steps to transcribe DNA into RNA. (2) Cas12a’s trans-DNase and trans-RNase activities can be initiated by CRISPR-Cas13a crRNA:DNA heteroduplexes. Furthermore, the wild-type Cas12a protein exhibits activatable trans-RNase activity, which conflicts with the trans-RNase activity of Cas13a. These interactions render the combination of Cas12a and Cas13a unsuitable for a single-tube reaction system. To confirm this concern, we combined the reaction components of both the CRISPR-Cas12a and Cas13a systems, including the Cas proteins and their specific crRNAs, into a single reaction tube (Supplementary Fig. 8). In experiments limited to Cas12a’s DNA target, without Cas13a’s RNA target, cleavage of the RNA reporter (FAM-rUrUrUrUrUrUrUrUrU-BHQ1) was observed, resulting in detectable fluorescent signals (Supplementary Fig. 8c). This confirms unintended activation of Cas12a’s trans-RNase activity, leading to false-positive indications of Cas13a’s RNA target presence. This experimental evidence further supports the conclusion that a single-tube approach combining the CRISPR-Cas12a and CRISPR-Cas13a systems for dual-gene detection is not feasible. (3) The trans-DNase activation of Cas12f1 is notably conserved compared to Cas12a. Despite its intrinsic RNase activity, mutating certain key residues inactivates this RNase activity while retaining trans-DNase functionality. Specifically, the Cas12f1/K173A mutant (drCas12f1) exhibited maximal reduction of its intrinsic RNase activity. Leveraging these findings, we devised a CRISPR-drCas12f1/Cas13a-based method for dual-gene detection (Fig. 6a). This approach employs CRISPR-drCas12f1 to target the human POP7 gene as an internal control, ensuring the validity of clinical sample collection and nucleic acid extraction methods. Simultaneously, the CRISPR-Cas13a system is utilized for the detection of the Hemagglutinin (H1) gene of the IVA/H1N1 virus in clinical throat swab specimens.
In the dual-gene CRISPR-based diagnostic strategy, we extracted nucleic acids from clinical throat swab samples and selectively pre-amplified the POP7 and H1 genes using specific primer through multiplex reverse transcription-recombinase polymerase amplification (RT-RPA). Subsequently, the amplified DNA products of POP7 and H1 were accurately detected using the CRISPR-drCas12f1 and CRISPR-Cas13a systems, respectively. Notably, while the CRISPR-Cas13a system integrates well with the RPA reaction30(Supplementary Fig. 9c, d), we observed that RT-RPA reagents inhibit the CRISPR-drCas12f1 system’s trans-DNase reaction rate and efficiency (Supplementary Fig. 9a, b). However, heating the RT-RPA reagents above 70°C for 1 minute before incorporating them into the CRISPR detection systems effectively mitigated these effects. Furthermore, both CRISPR-Cas12f1 and Cas13a systems exhibited a preference for single-stranded nucleic acids9, 12 (Fig. 1, Supplementary Fig. 10). To this end, during RT-RPA process, dsDNA targets were amplified using a phosphorothioate (PT)-modified primer to protect the target strand against T7 exonuclease degradation9. Following T7 exonuclease treatment in the CRISPR detection setup, the unprotected strand was degraded, leaving the ssDNA targets detectable by both drCas12f1 and Cas13a. We conducted detection using distinct fluorescent ssDNA (VIC-TTTTTTTTTTTT-BHQ1) and RNA (FAM-rUrUrUrUrUrUrUrUrU-BHQ1) reporters for drCas12f1 and Cas13a, respectively. The CRISPR-drCas12f1/Cas13a diagnostic approach resulted in four potential outcomes (Fig. 6b): (1) the absence of both target genes (POP7 and H1) resulted in no detectable fluorescence; (2) the presence of a single target gene triggered fluorescence emission from either the VIC or FAM reporter; (3) the simultaneous presence of both target genes enabled the concurrent detection of VIC and FAM fluorescence signals.
To evaluate the sensitivity of the CRISPR-drCas12f1/Cas13a system, serial dilutions of POP7 RNA and standard IVA/H1N1 virus RNA were employed. The system was capable of detecting as few as 40 attomoles of the POP7 gene and 200 copies per µL of the IVA/H1N1 virus (Fig. 6c). In testing specificity, RNA from other influenza A virus subtypes (H3N2, H5N1, H7N9) and influenza B viruses (Yamagata, Victoria) was subjected to analysis, observing no cross-reactivity (Fig. 6d). Clinical efficacy was validated by analyzing viral RNA extracted from 30 throat swab samples. The internal control gene, POP7, was detected in all 30 clinical samples (Fig. 6e). Importantly, the system accurately identified all 25 RT-qPCR positive samples (Ct values ranging from 20.1 to 38), while the 5 RT-qPCR confirmed negative samples showed no detection (Fig. 6f, Supplementary Fig. 11). Given the system’s accurate identification of all 30 clinical samples, the CRISPR-drCas12f1/Cas13a-based dual-gene detection method is very reliable and effective in detecting the IVA/H1N1 virus.