Test of Cas12a

Results

This section presents the results of activity tests performed with Cas12a.

As described in “Production”, we produced and purified two Cas12a variants (AsCas12a and LbCas12a). Their activity was tested by cleavage of linearized Pseudomonas putida KT2440 target DNA, which was analyzed by agarose gel electrophoresis. P. putida DNA was chosen due to its ease of cultivation in the lab and the well-characterized genome sequence. As shown in Figure 2, both Cas12a variants successfully cleaved the target DNA.

To assess activity rates in a high-throughput format, Cas12a activity was tested using a fluorescence reporter assay. The activity of the in-house produced variants was compared to a commercially available LbCas12a (Figure 3 A).

AsCas12a (Figure 3 C) showed strong activity, resulting in a pronounced fluorescence signal. However, the control reaction containing AsCas12a and crRNA only (without target plasmid) also produced high activity, suggesting nonspecific activation of the enzyme by the crRNA alone.

In contrast, LbCas12a (Figure 3 B) exhibited lower overall activity but greater specificity. A fluorescence signal was observed only in the presence of target plasmid DNA, while no activation occurred in reactions containing crRNA alone. Both the in-house produced LbCas12a and the commercial enzyme displayed comparable activities, confirming that the in-house preparation was of sufficient quality for further experiments.

Previous reports [1] have suggested that nonspecific Cas12a activity may be induced by the presence of magnesium ions. In our experiments, however, AsCas12a displayed nonspecific activity independently of Mg²⁺ concentration, whereas LbCas12a remained inactive in the absence of its specific target. Based on these observations, all subsequent experiments were conducted exclusively with LbCas12a

The detection sensitivity of the reporter system was evaluated using serial dilutions of plasmid DNA carrying different PAM-sequence targets. Differences in sensitivity were observed between the two targets. For target P1 (Figure 4 A), the signal could only be distinguished from the negative control at concentrations ≥0.2 nM, whereas for target P2 (Figure 4 B), activity was detectable down to 0.02 nM. These results demonstrate that the crRNA–target pair strongly influences detection sensitivity and suggest that further optimization will be required, as DNA concentrations in real biological samples may be considerably lower.

The sensitivity of the Cas12a–crRNA complex was evaluated using genomic DNA from P. putida. Two crRNAs were tested individually and in combination across different concentrations of genomic DNA and crRNA (Figure 5). Consistent with the plasmid-based experiments (Figure 4), crRNA P2 displayed stronger activity, underscoring that crRNA target selection is a critical determinant of Cas12a assay sensitivity.

To evaluate assay performance on more complex biological samples, DNA was extracted from potato tissue, including both uninfected plants and plants infected with Arsenophonus phytopathogenicus. Several crRNA targets were tested, but no activation above the negative control (Cas12a + crRNA only) was observed. This lack of detection may be due to a low abundance of Arsenophonus phytopathogenicus DNA in the extracts or suboptimal target selection. Further optimization will be required to improve detection sensitivity and specificity in such complex samples. DNA extracts from infected potato tissue were kindly provided by BIOREBA (Rheinach, Switzerland).

Material and Methods

Base Test Fluorescence Reporter

Fluorescence reporter assays based on Cas12a (Figure 1) were conducted to evaluate nuclease activity under varying buffer and target conditions. Cas12a enzymes (in-house produced AsCas12a and LbCas12a, and a commercial variant of LbCas12a) were tested in parallel. A ssDNA reporter labelled with a fluorophore and quencher was used to monitor cleavage activity (Cas12 ssDNA Reporter, SignalChem Diagnostics, Richmond, Canada).

General Assay Procedure

Fluorescence reporter assays were performed to monitor Cas12a nuclease activity. Reactions were assembled in 96-well clear plates (Greiner) with a total volume of 100 µL. Each assay contained Cas12a enzyme, the corresponding crRNA, target DNA, and a single-stranded DNA reporter labelled with a fluorophore and quencher (Figure 1). Unless otherwise specified, final concentrations were 2.5 µM Cas12a, 300 nM crRNA, 500 nM reporter, and 2 nM target DNA. Reactions were conducted in 1X Cas12 reaction buffer (1 M NaCl, 50 mM MgCl2, 200 mM HEPES, 1 mM EDTA, pH 8). Fluorescence (485 nm excitation; 520 nm emission) was monitored every 5 min at 30 °C for up to 16 h. Negative controls lacking Cas12a, crRNA, or target DNA were included in each experiment.

Plasmids used in this study:

Table 1: List of plasmids carrying different target DNA sequences for P. putida and A. phytopathogenicus used for the test of Cas12a.

Used crRNA sequences:

Table 2: List of crRNA targeting different DNA sequences for P. putida and A. phytopathogenicus used for the activation of Cas12a.

Enzyme Source Comparison

In-house purified Cas12a enzymes (AsCas12a, LbCas12a) were compared to commercial enzyme preparations. Protein concentrations were adjusted to match assay requirements. Dilutions were made where stock concentrations were limiting. Identical plasmid targets and reporter concentrations were used for both enzyme sources.

Plasmid Dilution Series

Purified plasmids (pIGEM25_007 and pIGEM25_008) were used as target DNA in place of synthetic PAM sequences. Plasmid concentrations were determined with a NanoDrop spectrophotometer and converted to molarity based on sequence length. Both circular and linearized plasmid preparations were tested. Tenfold serial dilutions were prepared to final concentrations ranging from the nanomolar to the femtomolar range. Reactions were performed in triplicate using LbCas12a according to the general assay procedure to determine the detection limit and dynamic range.

Genomic DNA of Pseudomonas putida KT2440

Genomic DNA was extracted from Pseudomonas putida KT2440 glycerol stocks using the Monarch Spin gDNA Extraction Kit (Monarch, #T3010S) according to the manufacturer’s protocol for Gram-negative bacteria, with minor adjustments. Two biological replicates (two different clones) were processed. Cell numbers were estimated from optical density measurements at 600 nm, assuming 1 OD₆₀₀ corresponds to 10⁹ cells/mL. Appropriate volumes of cryostock were thawed on ice, pelleted by centrifugation at 21’000 g for 1 min, and resuspended in 90 µL PBS (pH 7.4). Cells were treated with 10 µL of lysozyme solution (25 ng/µL in dH₂O) and incubated at 37 °C for 10 min. Lysis was continued by adding 100 µL Tissue Lysis Buffer, followed by 10 µL Proteinase K and incubation at 56 °C for 45 min under agitation (1’400 rpm). After clarification of the lysate, 3 µL RNase A was added and samples were further incubated at 56 °C for 5 min. DNA was then bound to purification columns by addition of 400 µL Binding Buffer and sequential centrifugation steps (1’000 g for 3 min, followed by 21’000 g for 1 min). Columns were washed twice with 500 µL Wash Buffer and centrifuged at 21’000 g for 1 min each. DNA was eluted in 100 µL preheated Elution Buffer (60 °C) and centrifuged at 21’000 g for 1 min. Extracted genomic DNA was quantified on a NanoDrop spectrophotometer. Samples were stored at –20 °C until further use. Purified genomic DNA was subsequently introduced into Cas12a fluorescence reporter assays under the same reaction conditions established for plasmid DNA targets. To evaluate target recognition, two guide RNAs were tested individually as well as in combination. This approach allowed assessment of both single-site and multi-site targeting within the genomic DNA context. Appropriate negative controls lacking Cas12a, crRNA, or target DNA were included for each condition to validate assay specificity.

ARSEPH-Infected Potato Samples

Samples derived from potato tissue showing symptoms of Arsenophonus phytopathogenicus infection (provided by BIOREBA, Rheinach BL, Switzerland) were tested as additional targets in the Cas12a fluorescence reporter assay. crRNAs were designed against annotated Arsenophonus phytopathogenicus genomic regions, and corresponding plasmid constructs carrying these loci were generated to provide defined positive controls. DNA concentrations from the infected potato extracts and the plasmid constructs were determined spectrophotometrically. Tenfold dilution series were prepared following the same procedure established for the pIGEM plasmids. Cas12a reporter assays were then carried out with these samples according to the general assay procedure. In each experimental set, negative controls lacking Cas12a, crRNA, or target DNA were included to verify assay specificity.

Notebooks

In this section, the Notebooks are uploaded.

• Notebook Production May (PDF)
• Notebook Production June (PDF)
• Notebook Production July (PDF)
• Notebook Production August (PDF)
• Notebook Production September (PDF)

References

[1] B. Li u. a., „CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides“, Mol Ther Nucleic Acids, Bd. 19, S. 1043–1052, Jan. 2020, doi: 10.1016/j.omtn.2019.12.038.