Engineering
As the goal was to develop a Cas12a-activated test for the detection of plant pathogens, the application of engineering principles was crucial to our project. Beyond creating a first functional prototype, we aimed to build a modular and iterative system in which each subteam could independently apply the engineering cycle (Design, Build, Test, Learn) to its respective part, while contributing to the integration of a complete detection assay.
Given the project’s complexity, we compartmentalized our work into three main engineering modules:
Cas12a Production and Verification
Developing a reliable and cost-effective in-house production process for functional Cas12a enzymes.
Cas12a Target Activation and Detection
Optimizing reaction conditions, buffer systems, and guide RNAs to ensure robust and specific fluorescence activation upon target recognition.
Visual Readout Development
Engineering a gold nanoparticle based colorimetric readout as a portable, instrument-free detection system for field testing.
Each module followed the engineering cycle, allowing us to iteratively refine our design through data-driven learning. Below, we describe how Design, Build, Test, and Learn principles were applied in each module.
in-house Cas12a Production
Design
To make the detection system scalable and low-cost, we designed a workflow for in-house Cas12a production. Based on published expression systems[1], we planned to express three Cas12a variants AsCas12a, LbCas12a, and FnCas12a in E. Coli Rosetta (DE3). The goal was to produce functional protein with sufficient purity for diagnostic assays, without the need for expensive commercial enzymes.
Build
Each Cas12a variant was cloned in expression plasmids obtained from Addgene and transformed into E. Coli Rosetta (DE3). Protein expression was induced with IPTG, and cells were lysed by sonication.
Purification was achieved via immobilized metal affinity chromatography (IMAC), followed by buffer exchange into HEPES-based storage buffer. Protein concentration and purity were verified by NanoDrop and SDS-PAGE.
Test
SDS-PAGE showed clear bands at the expected molecular weight (~150–160 kDa), confirming successful expression and purification. Functional testing in downstream assays verified nuclease activity for both AsCas12a and LbCas12a, demonstrating that the simplified purification protocol produced active enzyme.
Learn
We found that additional purification steps (e.g., His-tag cleavage or further chromatography) were unnecessary, the partially purified enzymes were already active in detection assays. Cost analysis showed that in-house production reduced enzyme costs by >99 %, from ~5.30 CHF per reaction to ~0.013 CHF, enabling affordable large-scale testing.
The protocol will serve as a foundation for future improvements, such as higher yield expression or His-tag reuse in immobilization-based formats.
Cas12a Target Activation and Detection
Design
We designed fluorescence-based reporter assays to validate Cas12a functionality and establish optimal reaction conditions for sensitive and specific target detection. Each reaction contained Cas12a, a crRNA targeting a defined sequence, a double-stranded DNA target, and a single-stranded reporter oligo.
The most important part of this design stage was the selection and design of the crRNAs, positioned near PAM sites in the target organism. These crRNAs were first tested with pre-designed plasmid targets to confirm their ability to activate Cas12a through fluorescence readout. Once effective crRNAs were identified, the system complexity was gradually increased: first using genomic DNA extracted from Pseudomonas putida , and later DNA samples from Bioreba containing A. phytopathogenicus target DNA. This progressive testing allowed us to validate crRNA performance step by step, from controlled plasmid conditions to realistic biological samples.
To systematically optimize the assay, we planned experiments varying in different conditions and Cas12a variants and more.
Build
We constructed test reactions using 96-well plate setups for reproducibility and high-throughput data collection on a plate reader. Buffer systems were optimized to mimic commercial formulations (100 mM Tris-HCl, 500 mM NaCl, 100 mM MgCl₂) but adapted to our available reagents. The final working buffer was adjusted to pH 8, ensuring compatibility with the fluorescence reporter.
Each setup was run in duplicate or triplicate with defined positive and negative controls.
Test
Initial endpoint measurements confirmed fluorescence activation with AsCas12a, while LbCas12a showed weaker signal under identical conditions. Kinetic assays over 8–16 h revealed that AsCas12a efficiently cleaved the fluorescent reporter, even at very low target concentrations. However, further studies indicated that the AsCas12a had non-specific activations, which makes it not suitable for a sensitive test. In later tests the assay was only run for 2 hours as it was sufficient to get a strong enough signal if the Cas was activated.
Learn
From this engineering cycle, we identified LbCas12a as the most reliable enzyme for our detection system. Although AsCas12a exhibited higher apparent activity, this was due to its non-specific collateral activation, which initially misled our early testing results.
Visual Readout Development
Design
To translate the fluorescence-based system into a field-deployable test, we designed a colorimetric readout using gold nanoparticles (AuNPs) functionalized with thiol-linked oligonucleotides.
The intended mechanism was that successful Cas12a activation (and thus target recognition) would cleave linker DNA, release aggregated gold nanoparticles and create a visible red color, while a negative sample would keep gold nanoparticles aggregated and thereby at a blue/purple color.
Build
We prepared AuNP–SH-oligo conjugates by attaching thiolated ssDNA to 10 nm and 60 nm AuNPs via TCEP-mediated reduction. Several conjugation strategies were tested:
- Excess TCEP relative to ssDNA (standard method)
- Excess ssDNA relative to TCEP (literature-based method)
- Modified protocol with ethanol washing to remove excess reagents
Following conjugation, salt-aging was performed to stabilize the DNA–AuNP complexes. Samples were washed by multiple centrifugation and buffer exchange steps to remove unbound DNA.
Test
Binding between AuNP–DNA conjugates and complementary linker strands was evaluated by measuring absorbance at 520 nm and 560 nm, monitoring the color shift upon aggregation.
Initial tests revealed precipitation and color differences between samples, indicating variability in conjugation efficiency. Re-optimization of TCEP ratios and salt-aging steps improved the stability and reproducibility of the AuNP–DNA constructs.
Learn
The extended salt aging duration proved essential to achieve clean conjugation and reduce unwanted aggregation. We learned that centrifugation conditions (speed, temperature) strongly affect nanoparticle stability, suggesting the need for gentler handling during the next iteration.
The optimized conditions now provide a solid base for integrating the Cas12a cleavage step to trigger visible color change in future cycles.
Conclusion
By compartmentalizing our project into these three engineering modules, each subteam could efficiently apply the Design–Build–Test–Learn cycle.
This modular strategy allowed us to iteratively improve individual components while maintaining a unified goal, developing a low-cost, Cas12a-based, field-deployable detection system for plant pathogens.
Our current prototype successfully combines in-house enzyme production, sensitive molecular detection, and a visual output concept, providing a strong foundation for further refinement and real-world application.
References
[1]Purification of CRISPR-Cas12a/Cpf1’, Bio Protoc, vol. 8, no. 9, p. e2842, May 2018, doi: 10.21769/BioProtoc.2842.