Cas12a Overview
Also known as CRISPR-Cas12a, the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) system is an emerging mechanism used in point-of-care diagnostics such as at-home Covid testing. (Ganbaatar & Liu, 2021; see Cas12a DETECTR). CRISPR-Cas12a utilizes a selective endonuclease complex that exhibits collateral cleavage activity upon recognition of the target DNA, resulting in a detectable output (see Fig. 1).
Cas12a Justification
Although Recombinase Polymerase Amplification (RPA) produces large quantities of DNA amplicons, RPA can potentially lead to non-specific amplification. Because LANCET still required a means to detect specific nucleic acid sequences, we leveraged the programmable recognition ability of the CRISPR-Cas12a system to selectively detect our desired product, allowing us to convert DNA into either a fluorescent output measurable by a plate reader or a visual band on a Lateral Flow Assay (LFA).
Cas12a
The CRISPR family contains several variants of the Cas protein, each with different functions and uses. Cas9, for example, is widely established and used for genome editing, while Cas13 is a more recently characterized protein, utilizing the ability to target RNA in diagnostic tools such as SHERLOCK. Cas12a has the benefit of both specifically targeting target DNA and collateral cleavage, the ability to indiscriminately cut nearby ssDNA. These two functions make Cas12a the optimal choice to integrate into the LANCET diagnostic workflow, alongside both PDL and RPA (see Table 1).
| Feature | Cas9 | Cas13 | Cas12a |
|---|---|---|---|
| Targets | DNA | RNA | DNA |
| PAM site | NGG (Gleditzsch et al., 2019) | None (Rahimi et al., 2024) | TTTV (Ai et al., 2025) |
| Cleavage type | Single cut, no collateral cleavage (Li et al., 2023) | Collateral cleavage of surrounding RNA (Rahimi et al., 2024) | Trans-cleavage activity; Collateral cleavage of ssDNA (Rahimi et al., 2024) |
| Sensitivity | Limited for signal amplification (Wu et al., 2014) | Requires conversion from DNA to RNA | High sensitivity and specificity |
Table 1. Comparison of characteristics between Cas enzyme variants.
crRNAs
A crRNA, or CRISPR RNA, is a small 20-44 nucleotide RNA molecule that guides Cas proteins. Guide RNAs for Cas12a are composed of a scaffold region and a spacer region. The scaffold region is conserved across different crRNAs, and functions to help position and bind the sequence to the endonuclease. The spacer region is complementary to the target DNA sequence and serves to direct the endonuclease to the correct site for cleavage (Teng et al., 2019; see Fig. 2).
Cas12a Design
Cas12a Target Construct Design
To design a target DNA construct that would function optimally in our experimentation, we ensured our synthetic target sequence (BBa_25F7MSFN) was identical to the product produced by proximity-dependent ligation (PDL) and amplified by RPA.
crRNA Design
crRNAs for Cas12a diagnostics consist of two specific RNA regions: the scaffold region, making up the first 20 base pairs, and the spacer region, making up the following 20–24 base pairs. The scaffold region is a universal sequence positioned upstream of the spacer region in the Cas12a guide RNA construct. We utilized a scaffold region employed in studies by Li et al. (2022), Broughton et al. (2020), and Yan et al. (2023), which was also recommended by SignalChem Diagnostics.
The spacer regions are designed to be complementary to the Cas12a target DNA sequence adjacent to an upstream ‘TTTV’ protospacer adjacent motif (PAM) site. For our positive control experiments, we selected the crRNA and target DNA sequences used in a study by Li et al. (2022), which focused on the diagnosis of Mycoplasma pneumoniae.
For LANCET, crRNA and target DNA were designed using Benchling software (Benchling, 2025). Their platform enables us to generate guide sequences from our target DNA and predict off-target effects for each guide (see Fig. 3; see Engineering Success). crRNAs were designed to be complementary to a region within all RPA amplicons while minimizing off-target effects.
Fluorophore Quencher (FQ) Reporter
The ssDNA FQ reporter probe used in plate reader assays is composed of a 6-carboxyfluorescein (6-FAM) label at the 5’ end and a Black Hole Quencher 1 (BHQ-1) label at the 3’ end. The 6-FAM label fluoresces at a wavelength at 520 nm, while the BHQ-1 label absorbs wavelengths between 480 and 580 nm. In close proximity, the BHQ-1 label functions to suppress fluorescence emitted by the 6-FAM label. However, once the fluorophore is physically separated from the quencher, 6-FAM is able to fluoresce freely and produce a quantifiable signal in a plate reader.
To monitor Cas12a activity, we employed an FQ reporter to produce a measurable fluorescence signal upon cleavage. Collateral cleavage activity exhibited by an activated Cas12a cuts the reporter probe, allowing 6-FAM to freely fluoresce (see Fig. 4). The expressed fluorescence can then be quantified using a plate reader to determine the presence of the target DNA.
Lateral Flow Assay (LFA) Design
A Lateral Flow Assay (LFA) is a paper-based test-strip known for quick run time and easy result visualization. Rather than requiring expensive laboratory equipment such as a plate reader to measure fluorescence, LANCET utilizes an LFA for cheap on-site testing.
6-FAM Biotin (FB) Reporter
The FAM-Biotin reporter probe is a single-stranded DNA sequence, tagged with a 6-FAM label at the 5’ end and a Biotin label at the 3’ end (see Figs. 5-6). Similar to the FQ reporter, the FB reporter is cut by activated Cas12a, allowing for visual quantification in the presence of target DNA. However, in contrast with the FQ reporter, the FB reporter is utilized in an LFA, rather than a plate reader (see Fluorophore Quencher (FQ) Reporter).
Sample Pad
The Cas12a reaction is first performed in a tube before coming in contact with an LFA. After the reaction takes place, the mixture is absorbed by the sample pad. The sample pad has several key functions:
- Controlling liquid dispersion across the LFA
- Filtering large particles or inhibitors
- Pretreating with chemicals to prepare the sample for interaction with the rest of the test strip
Gold Nanoparticles
Once added to the sample pad, the reaction mixture first flows through the conjugate pad. This pad contains gold nanoparticles (AuNPs) which are used as the visual reporter in the LFA, appearing as red lines when they bind to the strip.
Our AuNPs are conjugated with anti-6-FAM mouse antibodies, which specifically recognize and bind to the 5’ 6-FAM tag on the reporter probe. The solution continues to flow through the test strip, with all gold nanoparticles bound to reporter probes (see Fig. 7).
Capture Antibodies
Streptavidin and anti-mouse goat antibody are capture antibodies present at the control and test line, respectively. When capture antibodies bind to antigens flowing through the assay, the AuNPs attached to 6-FAM form a red line, creating a visual line on the LFA.
Control Line
Streptavidin enables specific binding of the 3’ biotin tag on the reporter probe. When uncleaved FB reporter probes flow through the LFA, AuNPs form a visual output on the control line. Since all the FB reporters bind to the streptavidin, an insignificant number of gold nanoparticles flow to the test line, resulting in no visual line formation (see Cas12a with LFA).
Test Line
Anti-mouse goat antibody, which recognizes anti-6-FAM mouse antibodies, is the capture antibody on the test line. When the FB reporter is cleaved, 6-FAM labels which are bound to AuNPs are able to flow to the test line and form a visual result (see Fig. 8).
For a positive result, once Cas12a binds to the target sequence, it indiscriminately cleaves the nearby ssDNA FB reporter. The reaction mixture is then put pipetted onto the sample pad of the LFA, where the mixture flows to the conjugate pad and 6-FAM labels bind to AuNPs. The mixture continues to flow across the LFA to the control and test strips. The biotin binds to the streptavidin control line, whereas the 6-FAM labels bound to AuNPs flow through the strip and are captured by goat anti-mouse antibodies, creating a colored result only at the test line.
Cas12a Experimentation
Our Cas12a experimentation was divided into several workflows. We first confirmed the competency of our Cas12a reagents by running reactions with target DNA and crRNA sequences that were characterized in past literature. For LANCET-specific testing, we began experimentation with our synthetic target construct and later integrated Cas12a with PDL and RPA to validate the functionality of the Cas12a-LFA system in our overall diagnostic (see Cas12a Target Construct Design). All final reactions, including controls, were run in triplicates to ensure that results were reproducible and statistically significant.
Cas12a Positive Control Testing
Before testing with our own crRNAs, we ensured that the reaction performed as expected by using a validated crRNA sequence. After ordering the crRNA (BBa_25OC05KU) and corresponding target DNA (BBa_250WTFLC) from IDT along with Lba Cas12a enzyme and buffer from New England Biolabs, we used an optimized protocol from SignalChem Diagnostics to maximize reaction activity (New England Biolabs, 2021; see Table 2).
| Reagent | Stock Concentration Used | Volume of Stock Used | Final Concentration in Reaction |
|---|---|---|---|
| LbCpf1 | 1000 nM | 5 μL | 250 nM |
| Buffer | 10x | 2 μL | 1x |
| crRNA guide | 2000 nM | 2.5 μL | 250 nM |
| Target DNA | 800 nM | 6.25 μL | 250 nM |
| FQ Reporter | 4000 nM | 1.25 μL | 250 nM |
| Nuclease-free Water | - | 3 μL | - |
Table 2. Optimized protocol for concentrations and volumes of reagents utilized in the 20 μL Cas12a reaction.
Fluorescence from the reporter molecule was quantified using a plate reader, and reads were optimized for 6-FAM by setting the plate reader to an emission read of 528/20 and an excitation read of 485/20.
To assess the success and specificity of Cas12a’s collateral cleavage activity, we included multiple control reactions:
- No target DNA: The absence of target DNA ensured that collateral cleavage was not activated without specific recognition of the crRNA to its complementary sequence.
- No Cas12a enzyme: Because Cas12a exhibits collateral cleavage, a reaction without Cas12a should not produce fluorescence, as fluorescent signals should depend on enzymatic activity.
Initial attempts were unsuccessful, as background fluorescence at similar rates to the experimental groups were exhibited by the control reactions (see Fig. 9).
Troubleshooting FQ Reporter Probe
Since the high background fluorescence in our control reactions suggested the presence of a leaky reporter, we replaced the commercial probe from SignalChem Diagnostics with a custom FQ reporter from IDT. We verified that our new reporter did not exhibit a high baseline fluorescence by comparing the fluorescence of the reporter with a blank well (see Fig. 10).
Once we confirmed that our reporter was not faulty, we ran the full Cas12a reaction with our control reactions and quantified the results using our fluorescence plate reader (see Fig. 11).
With the new FQ reporter, our experimental sample with verified crRNA and target DNA produced a significantly higher fluorescent signal compared to the control reactions, indicating successful collateral cleavage functionality from Cas12a (see Fig. 11). We also ran a calibration curve for our plate reader using purified 6-FAM labels to correlate relative fluorescence units (RFU) to absolute values of 6-FAM probe concentration (see Fig. 12; see Cas12a Modeling).
Using the relationship between RFU and 6-FAM concentration, we were able to get a linear correlation that follows the equation RFU = 94.1 × [6-FAM] - 95.8 with an R² = 1, indicating a nearly perfect linear relationship between measured fluorescence and probe concentration. We arranged this equation to get [6-FAM] = (RFU + 95.8)/94.1, allowing us to convert experimental RFU into 6-FAM probe concentration and assess the collateral cleavage activity of the Cas12a system (see Fig. 13).
By converting RFU values into 6-FAM concentration, we saw that our experimental sample exhibited a steady increase in cleaved reporter concentration over time, while both control reactions (no target DNA and no Cas12a enzyme) maintained insignificant baseline fluorescence. This significant increase in the experimental sample confirmed that fluorescence resulted from activation of the Cas12a system through specific target DNA recognition and not from a degraded reporter probe or high background signal (see Fig. 13).
Cas12a with LFA
After validating fluorescent output using the plate reader, we tested the positive control Cas12a reaction on an LFA. Using LFA strips from SignalChem Diagnostics, we used a modified protocol with concentrations and volumes optimized for Cas12a-LFA specific experimentation (see Table 3; see Fig. 14).
| Reagent | Stock Concentration Used | Volume of Stock Used | Final Concentration in Reaction |
|---|---|---|---|
| LbCpf1 | 1000 nM | 12.5 μL | 250 nM |
| Buffer | 10x | 5 μL | 1x |
| crRNA guide | 2000 nM | 6.25 μL | 250 nM |
| Target DNA | 844 nM | 14.81 μL | 250 nM |
| FB Reporter | 4000 nM | 3.125 μL | 250 nM |
| Nuclease-free Water | - | 8.315 μL | - |
Table 3. Concentrations and volumes of reagents utilized in the 50 μL Cas12a-LFA reaction.
For experimentation with LFAs, we ran our experiments along a control reaction, containing all reagents except for target DNA, ensuring that the Cas12a enzyme remains catalytically inactive in the absence of specific target recognition by the crRNA.
The LFA produced an expected output, as the experimental sample applied to the LFA on the top showed successful separation of 6-FAM indicated by some AuNPs bound to the goat anti-mouse antibody coated test line (see Fig. 14; see Test Line). A negative reaction with no target DNA was applied to the LFA on the bottom, which showed a result only on the control line, indicating that 6-FAM was not cleaved and AuNPs remained bound near the streptavidin (see Fig. 14; see Control Line).
Although binding to the test line may be faint, our results demonstrate the visual difference between our experimental and negative samples, signifying that a non-significant number of 6-FAM bound AuNPs flow through the LFA past the control line when the Cas12a enzyme is catalytically active and can cleave the FB reporter (see Fig. 14).
Cas12a Target Construct Testing
Once we confirmed that our reagents were able to produce clear results with characterized sequences, we began testing with our own crRNA (BBa_252J5WRW) designed using Benchling and its corresponding target DNA construct (see Cas12a Target Construct Design; see crRNA Design). We used the optimized protocol from positive control experimentation and the same control reactions to validate performance of the Cas12a system with LANCET-specific sequences (see Fig. 15).
Results from the plate reader show a steady increase in cleaved FQ reporter concentration over time. The similarity of our results with LANCET-specific sequences compared to that of the verified crRNA show that our experimental sample was able to activate collateral cleavage of Cas12a and produce fluorescence. In comparison, the control reactions showed no significant fluorescence increase, showing consistent and reliable results for Cas12a experimentation.
Our integrated Cas12a reaction was also visualized on an LFA to confirm that our fluorescence-based results were consistent with a visual readout. Once again, the LFA functioned as expected, with the negative reaction only exhibiting a control line from biotin-streptavidin binding, while the experimental sample showed a visual at the test line indicative of successful cleavage of the FB reporter (see Fig. 16).
Unlike experimentation with the verified sequences where the band at the test line appeared faint, the reaction with Cas12a target construct produced a single distinct output, strongly suggesting successful cleavage of the FB reporter by Cas12a (see Fig. 16). The lack of a test line in the negative sample verifies Cas12a’s specificity in recognition of a target sequence and demonstrates the absence of nonspecific cleavage or undesired flow through of AuNPs.
Conjugating Cas12a with RPA
The final step of our experimentation was to ensure that Cas12a could be integrated with PDL and RPA within our overall diagnostic workflow. We followed the same experimental protocols as before and also included an additional negative control, consisting of mismatched target DNA to the crRNA to further ensure the specificity of our Cas12a system (see Fig. 17).
From conjugating Cas12a with the rest of the LANCET pipeline, we found significantly greater cleaved 6-FAM concentration for the experimental sample compared to the negative controls, indicating successful assay activity (see Fig. 17). Our results show that we were able to successfully induce Cas12a activity at a similar rate to the positive control with target DNA construct, demonstrating the compatibility of Cas12a as the end-stage quantification of our diagnostic workflow’s results (see Fig. 17).
Additionally, we administered our full PDL-RPA-Cas12a reaction on an LFA, which resulted in successful cleavage of the FB reporter as shown by the strong test line exhibited by the experimental sample (see Fig. 18). The negative sample only showed an output at the control line, confirming that collateral cleavage only occurred in the absence of target DNA (see Fig. 18).
The distinct visual difference between experimental and negative samples highlights the potential for LANCET to provide a rapid visual output that creates ease of point-of-care testing. The successful integration of the Cas12a-LFA system within our pipeline demonstrates LANCET’s ability to specifically detect the CspZ protein biomarker in low volume samples.
Cas12a Sensitivity Testing
After confirming our ability to produce valid results at ideal target DNA concentrations, we wanted to test the specificity of our Cas12a system at lower target DNA concentrations. Published literature has demonstrated Cas12a systems to be specific with target DNA concentrations as low as one picomolar (Huyke et al., 2022). To investigate this, we added three positive controls while testing for fluorescence in a plate reader, keeping all reagent concentrations the same while adjusting the final target DNA concentration to 100, 40, and 16 nM respectively (see Fig. 19). These concentrations are lower than our ideal concentrations, but still significantly higher than the limits of detection found in literature.
Our results are consistent with the expectation that lower target DNA concentrations correspond to lower fluorescence output. All four experimental reactions exhibit significant and quantifiable fluorescence expression, with the lowest concentration (16 nM) still producing fluorescence measurably higher than the negative controls. This proves LANCET’s ability to detect Lyme even at low target DNA concentrations possible in early stage detection.
Ongoing work includes testing the Cas12a-LFA reactions with amplified DNA from samples at expected protein concentrations across multiple stages of Lyme to discover the threshold for complete assay viability. Overall, our findings so far support LANCET as a promising novel diagnostic platform for Lyme disease with the potential to overcome current limitations to detection and improve clinical outcomes by enabling early, on-site testing.
Validating Predictions from CASPER
Our CASPER software is designed to generate high-scoring crRNAs and RPA primers based on target sequences. To test the accuracy of the generated crRNAs, we chose the highest scoring design based off of our RPA amplified target sequence. The CASPER generated crRNA was run with RPA amplified PDL product (see Fig. 20).
Our results show the CASPER crRNA exhibited significant collateral cleavage of the FQ reporter. When comparing CASPER with Benchling designed crRNAs, enzyme efficiency remains on-par, with similar levels of FQ reporter cleavage being exhibited.
