Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis

doi:10.21203/rs.3.rs-2586294/v1

Download PDF

Article

Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis

https://doi.org/10.21203/rs.3.rs-2586294/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 05 Mar, 2024

Read the published version in Nature Communications →

Version 1

posted

You are reading this latest preprint version

Trans-cleavage rates of Cas endonucleases such as Cas12a remain limited despite optimisation efforts. We found that Cas12a ribonucleoproteins poorly bind to, and are only minimally activated by small circular DNA nanostructures which partially match the gRNA sequence. However, activation is restored when the nanostructure is linearised. Building on this finding, we established a novel Autocatalytic CRISPR/Cas12a Amplification Reaction (AutoCAR) system which allows a single nucleic acid target to activate multiple ribonucleoproteins, and increase the achievable trans-cleavage rates per target by over 3 orders of magnitude. A rate equation-based model was built to interpret the observed nonlinear, near-exponential rate trends. The AutoCAR system was applied for genomic DNA (H. Pylori) and RNA (SARS-CoV-2) diagnostics at 1 aM level sensitivity (1.4 copies/µL and 0.36 copies/µL, respectively), comparable to the polymerase chain reaction without any additional signal amplification at room temperature. The RNA detection with Cas12a RNPs did not require reverse transcription.

Biological sciences/Biochemistry/Biocatalysis

Biological sciences/Biochemistry/DNA

CRISPR/Cas12a

DNA nanostructure

autocatalysis

Cas12a RNP

catalysis kinetics

nucleic acid detection

The unprecedented discoveries of CRISPR/Cas systems continue to transform growing areas of biomedicine, synthetic biology, and biotechnology^1–4. Guided by an RNA molecule (gRNA, crRNA or sgRNA⁵), programmable endonuclease activity of Class 2 CRISPR/Cas systems provides a versatile toolkit to design and build nucleic acid/protein structures for gene editing and beyond^6–10. In particular, Cas12a ribonucleoprotein (RNP), with its special characteristics of sequence-dependent cis-cleavage and sequence-independent trans-cleavage upon RNP activation¹¹, continues to be widely explored for biomolecular diagnostics. The key application areas include nucleic acid detection, where low cost CRISPR/Cas-based tests for non-specialist users can be developed^{10, 12, 13}. In these applications, the trans-cleavage enzymatic activity of Cas12a RNP is required to catalyse degradation of single strand DNA. However, its limited catalytic speed (with reported values from 0.07 to 7 turnovers per sec¹⁴) is insufficient to realize efficient nucleic acid detection without additional amplification strategies. Although various ideas have been introduced to increase the overall endonuclease trans-cleavage levels, such as gRNA modifications, sequence optimization¹⁵, trans-cleavage substrate modifications^{16, 17}, and chemical enhancers¹⁸, the overall catalytic efficiency of a single Cas12a RNP remains limited, which is challenging for certain application. More importantly, the conventional one-to-one correspondence between target molecules and activated RNPs limits the overall enzymatic activity level in CRISPR/Cas systems which hinders potential applications in and beyond diagnostics. Explorations of novel strategies to expand the enzymatic activation pattern are important to realise the full potential of Class 2 Cas enzymes.

Autocatalytic reactions in biochemistry, particularly those based on nucleic acids have been widely explored for applications for diagnostics¹⁹, biosensing²⁰, imaging²¹, gene editing²² and in synthetic biology^{23, 24}. Specific nucleic acid sequences have been designed which are able to form DNA circuits²¹, or realize alternative (e.g. DNAzyme-induced) feedback loops in autocatalytic reactions^{25, 26 27}. A recent exploration utilized such nucleic acid circuits to realize autocatalysis in a CRISPR/Cas biosensing system, which increased the sensitivity of DNA detection to the attomolar range without additional amplification¹⁹. This design¹⁹ relies on a commonly used nucleic acid strand replacement scheme based on the Watson-Crick pairing dynamics to close the circuits, while the release of free RNA molecules in the circuit leads to a potential risk of self-degradation of activated Cas12a RNPs²⁸. However, the interactions of designed DNA nanostructures with CRISPR/Cas12a endonuclease activity remains unexplored, and their autocatalytic potential has not yet been identified.

In this work, we report a hitherto undiscovered property of the Cas12a RNPs that the activation of its trans-cleavage can be restricted with very high selectivity by a special design of a small circular DNA nanostructure which partially matches the gRNA sequence. The trans-cleavage can be restored by the action of other activated Cas12a RNPs. Building on this finding we established a novel Autocatalytic CRISPR/Cas12a Amplification Reaction (AutoCAR) system, which is able to break the standard “one target - one Cas12a RNP” activation pattern^{8, 11, 29}, and produce a feedback loop for Cas12a RNP activation to generate trans-cleavage products at very high rates. We have shown that the increased output of trans-cleavage products is attributed to the increased number of Cas12a RNPs that were activated by our DNA nanostructures in addition to target activation, and the non-linear (near-exponential) trend of substrate cleavage products confirmed the presence of autocatalysis. A rate equation-based model of the AutoCAR system was established which allowed us to interpret the observed trans-cleavage rates. The AutoCAR system has been further applied for novel nucleic acid diagnostics where, for the first time, 1 aM sensitivity (~ 1 copy/µL) for DNA has been reached without any additional signal amplification at room temperature. A similar sensitivity (~ 1.4 copies/µL) was achieved for the detection of genomic DNA from a pathogen, H. pylori. Furthermore, as autocatalysis overcomes the limitation of low Cas12a trans-cleavage rates for RNA targets ²⁸, we have been able, for the first time, to demonstrate RNA detection with Cas12a RNPs alone, without reverse transcription and additional amplification schemes, and with unprecedented sensitivity of 1 aM, where we were able to identify 0.36 copies/µL SARS-CoV-2 genomic RNA. The AutoCAR approach entirely avoids the generation of DNA amplicons, a by-product of conventional nucleic acid diagnostics, which is commonly responsible for contamination and false positives. These results expand the boundaries of current CRISPR/Cas biotechnology and pave the way for its potential new applications.

1. Introducing the AutoCAR scheme

In a standard CRISPR/Cas12a activation scheme^{29, 30}, the Cas12a endonuclease protein binds to gRNA to form a functional RNP which scans whether the surrounding nucleic acid sequences present a A/T-rich PAM region and are complementary to the gRNA spacer region. Once a suitable target DNA has been found, the Cas12a RNP binds to it and forms a double strand structure (R-loop) between complementary sequences^{30, 31}. The resulting activation of Cas12a RNP trans-cleavage activity can indiscriminately degrade any presented single strand DNA (ssDNA) sequence (Fig. 1A). This endonuclease activity is typically evaluated by the cleavage and subsequent unquenching of ssDNA-linked quenched fluorescent reporters, and may be used to amplify the signal of nucleic acid detection^{8, 10}.

Our AutoCAR CRISPR/Cas12a activation scheme is centred around a specially designed small DNA nanostructure comprising a double strand (dsDNA) and a ssDNA region, termed “Cir-mediator”. The dsDNA region contains both a A/T-rich PAM sequence and a spacer sequence matching the gRNA sequence of Cas12a RNP (Fig. 1B). When the ssDNA region of the Cir-mediator is cleaved, the nanostructure changes its topology and adopts a linear shape. We unexpectedly found that for specific lengths, the original circular Cir-mediator structure and its linearized form produce significantly different Cas12a activation patterns. Hence, we hypothesized that upon a suitable initiation of the reaction (by at least one linear DNA molecule complementary to Cas12a gRNA), such Cir-mediators and their matching Cas 12a RNPs can set up an autocatalytic reaction network (Fig. 1C). Autocatalysis takes place because activated Cas12a RNPs preferentially cut the short ssDNA region of Cir-mediators, which transforms the Cir-mediator back to a linearized form. These newly formed linear dsDNA oligos matching the gRNA of the RNPs cause activation of further RNPs thus closing the autocatalysis loop of the AutoCAR system (Fig. 1C).

2. Small circular DNA targets induce negligible Cas12a activation

The interaction of nucleic acid targets with the Cas12a RNPs was first explored in a series of linear ssDNA (L-ssDNA) structures with different length whose sequences were complementary to gRNA in the Cas12a RNP (Fig. 2A) and they additionally included an A/T-rich PAM region^{10, 32}. We verified by UNAFold simulations³³ that these linear ssDNA oligos do not form complex secondary structures in the condition of our experiment (Figure S1). Consistently with the previously reported properties of Cas12a RNP trans-cleavage¹¹, when the total length of target ssDNA region was decreased from 27 nt to 15 nt, and the binding length of DNA to gRNA (blue region in Fig. 2A) decreased from 21 nt to 9 nt, the trans-cleavage rates of Cas12a RNP (measured with ssDNA-linked fluorescence quenched reporters) also decreased by over 90% (Fig. 2B). A closely similar but less effective Cas12a RNP activation pattern was also found for different length of dsDNA targets (Figures S2, S3). We then discovered that forming a circular ssDNA structure (Cir-ssDNA) from the target ssDNA oligo (Figs. 2C, S4-5) suppresses the Cas12a RNP activation even though the L-ssDNA from which it is formed is still able to produce effective activation (Figs. 2D, S6). The ratio of activation efficiencies between Cir-ssDNA and linear DNA was found to be smallest for target ssDNA length of 20 nucleotides, with only about 3% of Cas12a activation remaining when circular ssDNA nanostructures were used as a target (Fig. 2E). Although this 20 nt long Cir-ssDNA still yields a statistically higher fluorescence signal compared with negative control (~ 15% higher, Fig. 2F), it is much lower (~ 23 times lower) compared with L-ssDNA of the same length in a 90 min reaction (Fig. 2F). Increasing the Cir-ssDNA length from 20 nt to 27 nt progressively restores the RNP activation, while Cir-ssDNA with lengths shorter than 20 nt produces a considerably lower level of overall Cas12a activation even in their linear form (Figure S7). Therefore, 20 nt oligos (L-ssDNA and Cir-ssDNA) were selected for further investigations. In order to be able to control the trans-cleavage activity by using such circular DNA structures, a complementary DNA (c-DNA) oligonucleotide was bound to the 20nt Cir-ssDNA covering the PAM and gRNA-binding regions of the Cir-ssDNA forming a Cir-mediator (Method 4.2, Figure S8). Informed by earlier reports that trans-cleavage of Cas12a can effectively and indiscriminately degrade ssDNA backbones but not dsDNA ^{11, 29}, we verified by gel electrophoresis that the dsDNA region of the Cir-mediators was able to survive the exposure to Cas12a trans-cleavage without significant degradation. However, the same L-ssDNA sequence was significantly degraded after 60 mins of trans-cleavage digestion and failed to activate its corresponding Cas12a RNPs (Fig. 2G, S9). Finally, we characterized the Cas12a activation pattern of the Cir-mediator DNA nanostructures. We found a similar restriction of Cas12a activation by Cir-mediators as by Cir-ssDNA (Figs. 2H, S10).

3. Linear residues of Cir-mediators activate Cas12a RNP trans -cleavage

We have further tested whether the function of Cas12a RNP activation is restored by linearization of the Cir-mediators. To investigate this, the Cir-mediators were exposed to pre-activated Cas12a RNPs (expected to cut its single strand region), and then the products were applied for activation of its corresponding Cas12a RNP (Method 4.7). The results show that among all the DNA molecules exposed to trans-cleavage, only Cir-mediator (or its L-dsDNA form) was able to effectively activate its corresponding Cas12a RNPs, while for ssDNA molecules (Cir-ssDNA or L-ssDNA) similarly exposed to trans-cleavage the Cas12a RNP activation efficiency was significantly reduced (Fig. 2I, S11). In addition, with increasing concentration of trans-cleavage-treated Cir-mediators, the downstream Cas12a activation efficiency was also increased (Figure S12). Combined, these data confirm that Cas12a trans-cleavage is able to break the ssDNA region of the Cir-mediator structure, thus returning its dsDNA region to linearity, which then can serve as a new target for activation of further Cas12a RNPs.

4. Mechanism of restricted Cas12a activation by circular DNA structure

To further investigate the mechanism of the observed restricted activation and the interaction between the formed Cir-mediators and its corresponding Cas12a RNPs, fluorescence resonance energy transfer (FRET) assays were carried with the donor (D) Cy3 fluorophore at the 5’ end of gRNA, and an acceptor (A) Cy5 fluorophore on an internal dT nucleotide adjacent to the PAM region of the 20 nt target strand (Fig. 3A). The sequence location of the FRET acceptor in linear (L-ssDNA or L-dsDNA) and circular (Cir-ssDNA or Cir-mediator) molecules bound to gRNA is the same due to basic Watson-Crick complementarity. In comparison to the original linear ssDNA which is known to stably bind to corresponding Cas12a RNP, both Cir-ssDNA and Cir-mediators showed a significant (about 72%) decrease of the FRET signal which was only slightly higher (6%) than the background (no Cas RNP, only oligos) (Figs. 3B, S13). Since the overall reaction environment of Cas12a RNP tested with linear and circularized DNA was the same (Methods 4.5), we considered whether the FRET signal change could be attributed to the distance change between donor and acceptors as a result of binding of oligos to the RNP. This distance change is limited by the distance change taking place upon RNP activation. Molecular simulations by using PDB 3D Protein Feature View³⁴ were carried out (PDB, 5XUS = activated Cas12a RNP, 6NME = inactivated Cas12a RNP), to estimate the donor-acceptor distance in inactivated (3.564 nm) and activated (3.732 nm) Cas12a RNP. (Fig. 3C). Their difference of 0.168 nm is due to Cas12a RNP configuration change which takes place during RNP activation. The FRET efficiency in an activated Cas12a RNP observed here was around 86% (Figure S14), which corresponds to donor-acceptor distance of 3.99 nm, according to the relationship \({E}_{FRET}= 1/1+{\left(\frac{R}{{R}_{0}}\right)}^{6}\), where \({R}_{0}\) is the Forster distance for Cy3-Cy5 of 5.4 nm and \(R\) is Cy3-Cy5 distance ¹⁸. The 0.168 nm distance change of Cas12a protein configuration produces only 0.03% FRET efficiency changes, which is much less than the 72% decrease shown in Fig. 3B. Therefore, we conclude that the fluorescence signal changes in Fig. 3B were due to significantly reduced binding affinity of Cir-mediators to RNPs compared to their linearized form.

5. Cir-mediators increase the overall levels of trans -cleavage but not Cas12a cleavage rates

Having established that the Cir-mediators can be linearized by RNP trans-cleavage, a step which can then induce further Cas12a RNP activation, we set up an Autocatalytic CRISPR/Cas12a Amplification Reaction (AutoCAR) system (Fig. 4A). To this aim we combined two types of Cas12a RNPs loaded with two different gRNA sequences. Cas12a RNP1 with gRNA1 targeting the initial triggering DNA was used to initiate the reaction, while the autocatalysis loop employs Cir-mediators and Cas12a RNP2 with gRNA2 targeting the dsDNA region of the Cir-mediators. Short ssDNA linked fluorescent quenched reporters ⁷ served to monitor overall AutoCAR (Method 4.8). In the AutoCAR system Cas12a RNP1s are activated by the triggering DNA, hence their trans-cleavage cuts the surrounding ssDNA including the ssDNA region of Cir-mediators. A significantly higher fluorescence signal was found in the AutoCAR system, compared to a standard CRISPR/Cas12a reaction without Cir-mediators (Fig. 4B) which is indicative of elevated trans-cleavage level (7.3 times higher for 1 hour and 14.4 times higher for 2 hours reactions, in our conditions). Additionally, with increasing concentration of Cir-mediator in the AutoCAR system, the fluorescence signal at different corresponding times also increased (Fig. 4C, S15). These data indicate higher overall levels of trans-cleavage in the AutoCAR system than in a standard CRISPR-Cas reaction with the same amounts of reaction-initiating targets. Unlike the well-established standard CRISPR/Cas12a trans-cleavage activation scheme ¹⁸ which shows a linear correlation between reaction time and fluorescence intensity changes (Figure S16), the fluorescent signal in the AutoCAR system is able to non-linearly increase with reaction time (Figure S17). Furthermore, for the same concentrations of target DNA to activate CRISPR/Cas12a trans-cleavage with (AutoCAR scheme) or without (standard CRISPR/Cas12a reaction scheme) Cir-mediators, the trends for cleaved number of fluorescence reporters with reaction time varied significantly. The standard reaction showed the expected linear correlation but the AutoCAR scheme produced a super-linear, nearly exponential trend (Fig. 4D).

To rule out the possibility that this altered trans-cleavage trend is due to single Cas12a RNP trans-cleavage rates being modified by simply the presence of Cir-mediator molecules in the solution, similarly to earlier reports for other molecules ¹⁸, a standard Michaelis-Menten analysis was used ¹⁴. We note that such analysis assumes quasi-steady state conditions which is only approximately satisfied in limited time windows. Empirical estimates for trans-cleavage rates were deduced from fluorescence data from Fig. 4E, whose trends over limited time windows (0–6 mins = TW 1, 20–30 mins = TW 2) are close to linear. The Michaelis-Menten analysis applied in these time windows yielded closely similar estimates for the turnover rates K_cat = 0.08 ± 0.02 s^− 1 (Figure S18, Table S1), which were well aligned with literature values ¹⁴. This is consistent with Cas12a trans-cleavage rates being unaffected by the presence of Cir-mediators, hence pointing to activation of additional Cas12a RNPs as a reason for enhanced reporter cleavage.

6. Autocatalysis in Cir-mediator assisted Cas12a reaction AutoCAR system – theoretical model and comparison with experiment

In order to understand the detailed impact of Cir-mediators on the Cas12a activation in the AutoCAR system, we set up a model system of chemical kinetics rate equations expanding the previously reported approach in a standard Cas12a RNP reaction system ¹⁴ (Supplementary Note). This model introduces the autocatalysis loop for Cas12a as well as two separate catalytic loops for the reporters (Fig. 4A, Equations 1, 2 5–12). The model allows to establish, in the Briggs-Haldane approximation, that the observed reporter cleavage rate (in specific reaction condition) is proportional to the concentration of activated RNPs (Supplementary Note, Eq. 24). This supports the application of the Michaelis-Menten theory in defined time windows to estimate the observed trans-cleavage rates (Fig. 4E, S18) which align with published K_cat value as ~ 0.07 to ~ 0.09 s^− 1 for LbCas12a ¹⁴. The model also allows us to determine the approximate number of activated Cas molecules per single target molecule (R_{Cas12a/target}) (Figs. 4F, S16). We found that R_{Cas12a/target} in the presence of 20 nM Cir-mediators over the reaction time of ~ 1500 s has increased by 3 orders of magnitude (a factor of 1600 ± 500, Table S2). This means that, in contrast to a standard Cas12a reaction system where a single target DNA molecule activates a single Cas12a RNP in the reaction system, in AutoCAR a single target DNA leads to activation of large number of additional RNPs in the course of the reaction (Figure S19). Overall, the R_{Cas12a/target} showed a positive linear correlation with the concentration of Cir-mediators in the AutoCAR reaction system (Figure S21), which indicates an elevated numbers of activated Cas12a RNPs for same level of target nucleic acid sequence. The behaviour of the reporter cleavage rate (fluorescent signal) in other experimental conditions was also revealed, most notably in conditions when none of the reagents becomes depleted during reaction (Supplementary Note, Eq. 37) when near-exponential growth is predicted, in agreement with Figs. 4D and S17. Other experimental conditions, e.g. when Cir-mediators may become depleted but targets are negligible also result in a non-linear pattern of the reaction rate (Fig. 4F). Finally, once the Cir-mediators have been depleted, the reaction kinetics returns to the basic Michaelis-Menten model where the observed rates are constant and fluorescence signals continue to increase linearly with time, reflecting the cumulative effect of autocatalysis on the overall cleavage of the reporters, in agreement with Eq. 24 that predicts proportionality of the rates to the reporter concentration (Figs. 4E, 4F, S20). In the event that the reporters become depleted, the fluorescent signal saturates (Figure S22).

7. Cir-mediator assisted Cas12a autocatalysis applied to ultra-sensitive DNA and RNA detection

Enhanced trans-cleavage and near-exponentially increased fluorescence output obtained in the AutoCAR system are ideally suited for nucleic acid detection at high sensitivity. We found that AutoCAR was able to reveal the presence of target DNA at 1 aM (Figure S23, Method 4.8). This is impossible in a standard CRISPR/Cas12a DNA reaction, which only reaches ~ 10 pM level without an additional amplification strategy (Figure S23, S24, Method 4.5). AutoCAR could also distinguish the change of target DNA concentrations at extremely low levels between 1 to 10 aM (Figure S25). In experimental conditions described in Method 4.11, AutoCAR had a linear range of 3 orders of magnitude from 1 aM to 1 fM (Fig. 5A). Furthermore, to assess if AutoCAR can be applied for real pathogen detection, the extracted H. pylori whole genome DNA was used as a target to initiate the AutoCAR system with the gRNA sequence of Cas12a RNP1 corresponding to the H. pylori glm gene fragment (Method 4.11). The results confirm that AutoCAR is able to detect the presence of 1.4 copies/µL of H. pylori genome DNA in the tested sample at room temperature which is in the aM range (Fig. 5B). This detection sensitivity is comparable to the current gold-standard qPCR (Figure S26), but without producing amplicons or the need for temperature cycling.

Furthermore, despite the ability of Cas12a RNP to directly recognize RNA sequences ²⁸, direct RNA detection by Cas12a is impractical due to a significantly lower level of trans-cleavage activity compared to DNA targets (Figure S27); as a result published methods rely on the combination of reverse transcription and additional amplification strategies. Autocatalysis in our AutoCAR overcomes this limitation. Here, we demonstrated the possibility of using Cas12a endonuclease to directly detect RNA by the AutoCAR scheme without reverse transcription nor amplification. To this aim the reporter concentration in AutoCAR was increased (Method 4.13) and we also added DTT as a Cas12a “trans-cleavage enhancer” to increase the overall system fluorescence signal level (Figure S28, Method 4.13). In such conditions, AutoCAR was able to directly detect RNA, without reverse transcription, at a sensitivity down to 1 aM and with a linear range of 3 orders of magnitude for quantitative detection (Fig. 5C). We also realized genomic RNA detection using samples containing SARS-CoV-2 viral particles (Methods 4.13, 4.16). AutoCAR was able to detect the presence of the N-gene of SARS-CoV-2 viral genome RNA at the sensitivity of less than 1 copy/µL (360 copies/mL), which is, again, comparable to the currently using gold standard RT-qPCR approach (Figure S29, Method 4.15). In addition, the fluorescence signal of AutoCAR system could also be read by an alternative readout instrument (a real-time thermocycler) which achieved the same 1 aM nucleic acid detection sensitivity (Figure S30, Method 4.12).

The wide range of novel applications of CRISPR/Cas12a systems are generally predicated on pairing of single nucleic acid molecules and single Cas12a RNPs^{5, 11, 14} which makes it challenging to establish more sophisticated reaction networks capable of autocatalysis. A recent report established a nucleic acid autocatalytic circuit and a downstream Cas12a RNP applied only for the detection of DNA target¹⁹. The initiation of autocatalysis in this system relies on the release of unbound free gRNA due to reduced double strand binding, which is known to be unstable in uncontrolled reaction environments, and this free gRNA can also be cleaved by activated Cas12a trans-cleavage²⁸. In addition, the continuous release of free gRNA has the potential to disrupt the R-loop in activated Cas12a RNP³⁵, hence leading to the interruption of unspecific ssDNA degradation which is the key to realize autocatalysis, and potentially lead to the breakdown of the autocatalysis loop. In contrast, in the AutoCAR system, the autocatalysis loop is realized via the interaction between a specifically designed DNA nanostructure (Cir-mediator) and Cas12a RNP with its programmable enzymatic activity, where the above considerations do not apply. Once the reaction is initiated by at least a single targeted nucleic acid sequence, the Cir mediators recruit and activate large amounts of Cas12a RNPs without exposing free gRNA molecules. Following the accumulation of activated Cas12a RNPs (Fig. 4A, RNP1 and RNP2), the total cutting rate of the reporter continuously increases until either Cir-mediators or Cas12a RNP2 are depleted, which can lead to an exponential fluorescence increase pattern during the period of increasing Cas12a RNP2 activation.

The key property of Cas12a RNP which facilitates autocatalysis is its inability to bind and be-activated by Cir-mediators in their circular topology – which changes when the topology barrier is overcome by trans-cleavage, and they become linearized. The process of Cas12a RNP activation requires the unwinding of the double helix structure of the target DNA, which is known to be torsionally regulated³⁶. RNA-guided DNA recognition occurs by strand separation of a protospacer target to allow Watson–Crick base pairing between the DNA targeted strand (TS) and the spacer sequence of a gRNA, and the unwinding of a non-targeted strand (NTS)³⁷. After cis-cleavage, the Cas12a RNP remains bound to the PAM-proximal cleavage product and the RNP undergoes a conformational change enabling trans-cleavage. Thus the trans-cleavage process is predicated on the formation and dissociation of the R-loop - which requires torque³⁸. The Cir-mediators in AutoCAR are very short, corresponding to approximately one or two coils of the double-helix and about 7 nm long for the 20 nt Cir-mediator^{39, 40}. High torsional stress is expected due to small radius of curvature along the length of circle (shown by a purple arrow in Figure S32). Furthermore, the closed loop in the Cir-mediator makes it rotationally constrained, as the initiation of dsDNA unwinding in one location requires increasing of the winding in adjacent locations, and/or in the ssDNA region - unlike in a corresponding linear structure. Thus a topological barrier in the Cir-mediator prevents it from releasing torsional stress in the perpendicular direction (shown by an orange arrow in Figure S32). At the same time the Cir-mediators are too short to writhe or to supercoil which requires DNA length of ~ 20 nm or more⁴⁰. Because the Cir-mediator is expected to have a greater torsional stiffness to its corresponding linear form, it requires more energy to unwind and form the required R-loop structure between gRNA and target DNA for Cas12a RNP activation compared to topologically different standard L-dsDNA targets⁴¹. Thus we propose that bending and torsional stiffness⁴² provide intuitions as to possible reasons for reduced binding of Cir-mediators to the RNPs compared with the constituent linear dsDNA.

Trans-cleavage activity in CRISPR/Cas reactions underpins key applications of CRISPR/Cas biotechnology in bioanalysis, biomedical and diagnostic fields^{8, 43, 44}. Although the discovery of ortholog proteins with higher efficiency, or reengineering the protein, etc., can lead to incremental improvements, the limited trans-cleavage represents a challenge for expanding the applications of CRISPR/Cas-based biotechnologies, particularly in precise or Point-of-Care diagnostics^{12, 45, 46}. Establishing autocatalysis reaction networks provides a method to overcome this problem. In summary our AutoCAR approach represents a novel way to control the CRISPR/Cas12a enzymatic activity through autocatalysis which will expand the boundaries of CRISPR/Cas biotechnology and broaden its applications.

4.1 Materials

T4 ligase (NEB), T4 ligase buffer (NEB), exonuclease III (NEB), LbCas12a (NEB), NEB2.1 buffer (NEB), agarose (ThermoFisher), TBE buffer, Urea-PAGE (Bio-rad), SYBR Gold DNA dye (ThermalFisher), 100 bp DNA ladder (ThermoFisher), 10 bp DNA ladder (ThermoFisher), 6X DNA loading dye (ThermoFisher), DTT (ThermoFisher), QuantiNova SYBR Green PCR kit (QIAGEN, 208052), QuantiNova SYBR Green RT-PCR kit (QIAGEN, 208152), AMPLIRUN Helicobacter Pylori DNA control (Vircell, MBC049-R, NCBI No. NC_000915.1), deactivated SARS-CoV-2 viral particles (Certified Reference Materials, National Measurement Institute, Australia).

All DNA and RNA oligos are synthesized and modified by Sangon Bio-tech Ltd (Table S3).

4.2 Synthesis of the Cir-mediator DNA nanostructure

The Cir-mediator was synthesized in a three-step protocol (Figure S4): The Cir-ssDNA was prepared using a reaction mixture containing 2 µL of the linear ssDNA oligo (100 µM), 4 µL of linker ssDNA oligo (100 µM), 2.5 µL of T4 ligase (NEBuffer), 1 µL of T4 ligase buffer, and 43 µL of DI water. The cyclization reaction was allowed to proceed at 16℃ for 12 hours and then 65℃ for 10min, following by holding at 4℃. Then, the un-ligated linear ssDNA and the linker ssDNA was depredated with a mixture containing 1.5 µL of exonuclease III, 10 µL of Cir-ssDNA product in 40 µL of 1×NEBuffer 2.1 buffer, and incubation at 37℃ for 100 mins followed with deactivation of exonuclease III at 75℃ for 30 mins. Afterwards, 1:1 molar ratio of cleaned Cir-ssDNA was mixed with its corresponding cDNA at room temperature for 10 mins to form the Cir-mediator molecule.

4.3 Gel electrophoresis to verify the formation of circular DNA structures

The formation of circular DNA was verified using agarose gel electrophoresis. Briefly, 4% agarose gel in 1×TBE buffer was prepared with SYBR Gold DNA dye (1.5 µL 10,000X into 30 mL agarose gel). 10 µL of DNA circularization product was premixed with 2 µL 6X DNA gel loading dye and then loaded onto gel for electrophoresis. The electrophoresis was carried out for 60 min at a constant voltage of 80V. 1.5 µL of 10 bp DNA ladder was used for molecular weight reference. Gel images were visualized by using Gel Doc + XR image system (Bio-Rad Laboratories Inc., USA).

4.4 Urea-PAGE electrophoresis to verify the formation of circular DNA structures

The formation of circularized DNA was verified using urea-PAGE electrophoresis. Briefly, 10 µL of DNA circularization product was premixed with 2 µL 6X DNA gel loading dye and then loaded onto premade15% Mini-PROTEANR TBE-Urea Gel (Bio-Rad, 4566055). The electrophoresis was then carried out for 60 min at a constant voltage of 110V. 2.5 µL of 10 bp DNA ladder was used for molecular weight reference. Gel images were visualized by using Gel Doc + XR image system (Bio-Rad Laboratories Inc., USA).

4.5 Standard CRISPR/Cas12a trans-cleavage activation assay

1 µL of 100 µM LbaCas12a endonuclease (NEB, M0653T) and 5 µL of 20 µM gRNA was mixed at 3.6 mL 1× NEBuffer 2.1, followed by adding of 6 µL of 100 µM Texas Red quenched reporter. The prepared standard CRISPR/Cas12a reaction mixture was stored at 4℃ for future use. For each CRISPR/Cas12a trans-cleavage activation reaction, 10 µL 10 nM of nucleic acid (L-ssDNA, L-dsDNA, Cir-ssDNA or Cir-mediator) with complementary sequence of gRNA was added into 90 µL prepared reaction buffer. The reaction was carried out at room temperature, and the fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader (iD5 Spectramax, Molecular Devices, USA).

4.6 Validation of Cas12a trans-cleavage for ssDNA and dsDNA degradation

1 µL of 100 µM LbaCas12a endonuclease (NEB, M0653T) and 5 µL of 20 µM gRNA was mixed at 3.6 mL 1× NEBuffer 2.1, and followed with the adding of 1 µM non-trigger ssDNA or dsDNA oligo. Then, 10 µL of 1 µM trigger ssDNA was mixed with 90 µL of the prepared standard CRISPR/Cas12a reaction mixture. The reaction was set at room temperature, and each of 10 µL cleavage product from 0, 10, 20, 30, 40, 50, 60 mins was mixed with 2 µL of 6X DNA gel loading dye, and then loaded onto 4% agarose gel for electrophoresis at a constant voltage of 80V for 60 min. Gel images were visualized by using Gel Doc + XR image system (Bio-Rad Laboratories Inc., USA).

4.7 Re-linearization of Cir-mediators by Cas12a trans-cleavage for further Cas12a activation

1 µL of 100 µM LbaCas12a endonuclease (NEB, M0653T) and 5 µL of 20 µM gRNA was mixed at 3.6 mL 1× NEBuffer 2.1, and followed with the adding of 100 nM of prepared DNA oligo (linear dsDNA, linear ssDNA, Cir-ssDNA or Cir-mediator). Then, 1 µM of trigger ssDNA oligo was added to activate Cas12a trans-cleavage for 60 mins at room temperature. Afterwards, 10 µL of the trans-cleavage product was transferred into 90 µL of the standard CRISPR/Cas12a reaction mixture with gRNA for Cir-mediator, and set at room temperature for 60 mins. The fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader (iD5 Spectramax, Molecular Devices, USA).

4.8 Preparation of the final AutoCAR reaction mixture

3 µL of 100 µM LbaCas12a endonuclease (NEB, M0653T), 5 µL of 20 µM gRNA1 (for trigger DNA/RNA) and 10 µL of 20 µM gRNA2 (for Cir-mediator) was mixed at 3.6 mL of 1× NEBuffer 2.1 and followed with the adding of 12 µL of 100 µM Texas Red quenched reporter. Afterwards, the prepared Cir-mediator solution was mixed to a final concentration of 50 nM (can be varied between 5–50 nM) before use to form the final AUTOCAR reaction mixture. Then, 10 µL of different concentrations of trigger DNA (ssDNA or dsDNA) with a complementary sequence to gRNA was mixed with 90 µL of the prepared final AUTOCAR reaction mixture to initiate the reaction. The reaction was set at room temperature and the fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader (iD5 Spectramax, Molecular Devices, USA). Alternatively, Cir-mediator can be replaced by 50 nM of the same volume of 1× NEBuffer 2.1 for a comparison with a standard Cas12a reaction as per Method 4.6 above.

4.9 Autocatalysis trans-cleavage kinetic in AutoCAR

To assess the kinetics of the AUTOCAR trans-cleavage pattern, the final concentration of 20 nM of LbaCas12a endonuclease (NEB, M0653T), 10 nM of gRNA1 (for trigger ssDNA) and 10 nM of gRNA2 (for Cir-mediator) was mixed with 1× NEBuffer 2.1, along with a constant concentration of 5 nM of target ssDNA. In addition, the mixture contained different concentrations of ssDNA reporter (0.156, 0.313, 0.625, 1.25, 2.5, 5 µM). The reactions were conducted at room temperature and the fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader at 120 sec intervals (iD5 Spectramax, Molecular Devices, USA).

4.10 Different concentrations of Cir-mediator in AutoCAR

The final concentration of 20 nM of LbaCas12a endonuclease (NEB, M0653T), 10 nM of gRNA1 (for trigger ssDNA) and 10 nM of gRNA2 (for Cir-mediator) was mixed with 1× NEBuffer 2.1, along with a constant concentration of 1 pM of target ssDNA and 2.5 µM of reporter. In addition, the mixture contained different concentrations of Cir-mediator (0, 2.5, 5, 10, 20 nM). The reactions were conducted at room temperature and the fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader at 120 sec intervals (iD5 Spectramax, Molecular Devices, USA).

4.11 FRET Investigation of interaction of Cir-mediators and Cas12a RNPs

To prepare the FRET assay, the 5’ of Cas12a gRNA was labelled with a 5’-Cy3 as a donor, and an internal dT at the 3’ of ssDNA (either linear or circularized form) was labelled with a Cy5 as an acceptor (Table S3). In brief, the Cy5 labelled ssDNA oligo was firstly mixed with its cDNA to form dsDNA or Cir-mediator, then 5 µL of 1 µM of the prepared DNA oligo was mixed with 95 µL of 1×NEBuffer 2.1 containing Cy3 labelled gRNA loaded Cas12a RNP to a final concentration of 50 nM for both the DNA oligo and Cas12a RNP. The fluorescence signals were then collected by using a plate reader (iD5 Spectramax, Molecular Devices, USA) with Ex/Em of 520/666 nm.

4.12 AutoCAR for DNA diagnostics on a plate reader

3 µL of 100 µM LbaCas12a endonuclease (NEB, M0653T), 5 µL of 20 µM gRNA1 (for the target DNA sequence) and 10 µL of 20 µM gRNA2 (for the Cir-mediators) was mixed at 3.6 mL 1× NEBuffer 2.1 and followed by adding of 12 µL of 100 µM Texas Red quenched reporter. Afterwards, the prepared Cir-mediator solution was mixed to a final concentration of 50 nM to form the final AUTOCAR reaction mixture for DNA detection. Then, 10 µL of different concentrations of target DNA (ssDNA, dsDNA or H.pylori genome DNA) were mixed with 90 µL of the prepared final AUTOCAR reaction mixture. The reaction was set at room temperature for 1 hours, and the fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader (iD5 Spectramax, Molecular Devices, USA).

4.13 AutoCAR for RNA diagnostics on a plate reader

3 µL of 100 µM LbaCas12a endonuclease (NEB, M0653T), 5 µL of 20 µM gRNA1 (for target RNA sequence) and 10 µL of 20 µM gRNA2 (for Cir-mediator) was mixed at 3.6 mL 1× NEBuffer 2.1 and followed by adding of 120 µL of 100 µM Texas Red quenched reporter and 36 µL of 1M DTT. Afterwards, the prepared Cir-mediator solution was mixed to a final concentration of 50 nM before the use to form the final AUTOCAR reaction mixture for RNA. Then, 10 µL different concentration of target RNA (RNA or SARS-CoV-2 genome RNA) was mixed with 90 µL of the prepared final c-Car reaction mixture. The reaction was set at room temperature for 1-1.5 hours, and the fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader (iD5 Spectramax, Molecular Devices, USA).

Zhu, H.C., Li, C. & Gao, C.X. Applications of CRISPR-Cas in agriculture and plant biotechnology (vol 21, pg 661, 2020). Nature Reviews Molecular Cell Biology 21, 782-782 (2020).
Chertow, D.S. Next-generation diagnostics with CRISPR. Science 360, 381-382 (2018).
Broto, M. et al. Nanozyme-catalysed CRISPR assay for preamplification-free detection of non-coding RNAs. Nature Nanotechnology 17, 1120-+ (2022).
Gayet, R.V. et al. Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release. Nature Protocols 15, 3030-3063 (2020).
Liu, G.W., Lin, Q.P., Jin, S. & Gao, C.X. The CRISPR-Cas toolbox and gene editing technologies. Molecular Cell 82, 333-347 (2022).
Anzalone, A.V., Koblan, L.W. & Liu, D.R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nature Biotechnology 38, 824-844 (2020).
Manghwar, H., Lindsey, K., Zhang, X.L. & Jin, S.X. CRISPR/Cas System: Recent Advances and Future Prospects for Genome Editing. Trends in Plant Science 24, 1102-1125 (2019).
English, M.A. et al. Programmable CRISPR-responsive smart materials. Science 365, 780-+ (2019).
Knott, G.J. & Doudna, J.A. CRISPR-Cas guides the future of genetic engineering. Science 361, 866-869 (2018).
Kaminski, M.M., Abudayyeh, O.O., Gootenberg, J.S., Zhang, F. & Collins, J.J. CRISPR-based diagnostics. Nature Biomedical Engineering 5, 643-656 (2021).
Li, S.Y. et al. CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Research 28, 491-493 (2018).
Tang, Y.N. et al. The CRISPR-Cas toolbox for analytical and diagnostic assay development. Chemical Society Reviews 50, 11844-11869 (2021).
van Dongen, J.E. et al. Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities. Biosensors & Bioelectronics 166 (2020).
Ramachandran, A. & Santiago, J.G. CRISPR Enzyme Kinetics for Molecular Diagnostics. Analytical Chemistry 93, 7456-7464 (2021).
Fozouni, P. et al. Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. Cell 184, 323-+ (2021).
Rossetti, M. et al. Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters. Nucleic Acids Res 50, 8377-8391 (2022).
Liu, S.T. et al. Systematically investigating the fluorescent signal readout of CRISPR-Cas12a for highly sensitive SARS-CoV-2 detection. Sensors and Actuators B-Chemical 373 (2022).
Deng, F., Li, Y., Li, B.T. & Goldys, E.M. Increasing trans-cleavage catalytic efficiency of Cas12a and Cas13a with chemical enhancers: Application to amplified nucleic acid detection. Sensor Actuat B-Chem 373 (2022).
Shi, K. et al. A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics. Science Advances 7 (2021).
Gao, Y.H. et al. Enzyme-Free Autocatalysis-Driven Feedback DNA Circuits for Amplified Aptasensing of Living Cells. Acs Applied Materials & Interfaces 14, 5080-5089 (2022).
Wang, Y.S. et al. An Autocatalytic DNA Circuit Based on Hybridization Chain Assembly for Intracellular Imaging of Polynucleotide Kinase. Acs Applied Materials & Interfaces 14, 31727-31736 (2022).
Yi, Z.Y. et al. Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo. Nature Biotechnology 40, 946-+ (2022).
Zambrano, A. et al. Programmable synthetic cell networks regulated by tuneable reaction rates. Nature Communications 13 (2022).
van Nies, P. et al. Self-replication of DNA by its encoded proteins in liposome-based synthetic cells. Nature Communications 9 (2018).
Wang, H.M., Luo, D., Wang, H., Wang, F. & Liu, X.Q. Construction of Smart Stimuli-Responsive DNA Nanostructures for Biomedical Applications. Chemistry-a European Journal 27, 3929-3943 (2021).
Wang, H.M. et al. Construction of an Autocatalytic Hybridization Assembly Circuit for Amplified In Vivo MicroRNA Imaging. Angewandte Chemie-International Edition 61 (2022).
Wei, J. et al. A Smart, Autocatalytic, DNAzyme Biocircuit for in Vivo, Amplified, MicroRNA Imaging. Angewandte Chemie-International Edition 59, 5965-5971 (2020).
Li, J.C. et al. Discovery of the Rnase activity of CRISPR-Cas12a and its distinguishing cleavage efficiency on various substrates. Chem Commun 58, 2540-2543 (2022).
Chen, J.S. et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity (vol 71, pg 313, 2021). Science 371 (2021).
Jeon, Y. et al. Direct observation of DNA target searching and cleavage by CRISPR-Cas12a. Nature Communications 9 (2018).
Swarts, D.C. Making the cut(s): how Cas12a cleaves target and non-target DNA. Biochemical Society Transactions 47, 1499-1510 (2019).
Li, Y., Li, S.Y., Wang, J. & Liu, G. CRISPR/Cas Systems towards Next-Generation Biosensing. Trends Biotechnol 37, 730-743 (2019).
Markham, N.R. & Zuker, M. UNAFold: software for nucleic acid folding and hybridization. Methods Mol Biol 453, 3-31 (2008).
Burley, S.K. et al. RCSB Protein Data Bank: powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences. Nucleic Acids Res 49, D437-D451 (2021).
Stella, S. et al. Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity. Cell 175, 1856-+ (2018).
De Vlaminck, I. et al. Torsional regulation of hRPA-induced unwinding of double-stranded DNA. Nucleic Acids Research 38, 4133-4142 (2010).
Swartjes, T., Staals, R.H.J. & van der Oost, J. Editor's cut: DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a. Biochem Soc Trans 48, 207-219 (2020).
Naqvi, M.M., Lee, L., Montaguth, O.E.T., Diffin, F.M. & Szczelkun, M.D. CRISPR–Cas12a-mediated DNA clamping triggers target-strand cleavage. Nature Chemical Biology 18, 1014-1022 (2022).
Marko, J.F. Torque and dynamics of linking number relaxation in stretched supercoiled DNA. Phys Rev E Stat Nonlin Soft Matter Phys 76, 021926 (2007).
Gao, X., Hong, Y., Ye, F., Inman, J.T. & Wang, M.D. Torsional Stiffness of Extended and Plectonemic DNA. Physical Review Letters 127, 028101 (2021).
Mirkin, S.M. in eLS (
Bouchiat, C. & Mézard, M. Elastic rod model of a supercoiled DNA molecule. The European Physical Journal E 2, 377-402 (2000).
Demirer, G.S. et al. Nanotechnology to advance CRISPR-Cas genetic engineering of plants. Nature Nanotechnology 16, 243-250 (2021).
Zhang, Y.X., Wu, Y.P., Wu, Y.F., Chang, Y.Y. & Liu, M. CRISPR-Cas systems: From gene scissors to programmable biosensors. Trac-Trends in Analytical Chemistry 137 (2021).
Li, Y., Liu, L.Y. & Liu, G.Z. CRISPR/Cas Multiplexed Biosensing: A Challenge or an Insurmountable Obstacle? Trends in Biotechnology 37, 792-795 (2019).
Wu, H. et al. Versatile detection with CRISPR/Cas system from applications to challenges. Trac-Trends in Analytical Chemistry 135 (2021).

There is NO Competing Interest.

AutoCARSI.docx
Supplementary information for AutoCAR manuscript
nrreportingsummaryYLAutoCAR.pdf
Reporting Summary

Download PDF

Journal Publication

published 05 Mar, 2024

Read the published version in Nature Communications →

Version 1

posted

You are reading this latest preprint version

Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

1. Introducing the AutoCAR scheme

2. Small circular DNA targets induce negligible Cas12a activation

4. Mechanism of restricted Cas12a activation by circular DNA structure

6. Autocatalysis in Cir-mediator assisted Cas12a reaction AutoCAR system – theoretical model and comparison with experiment

7. Cir-mediator assisted Cas12a autocatalysis applied to ultra-sensitive DNA and RNA detection

Discussion

Materials and methods

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1