iGEM 2025 — Team fhnw

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# Notebook

Developing a rapid and filed-ready test:
The CRISPR Cas12a detecting Arsenophonus

## Production and Purification

* Preparation
* Protein Expression
* Purification

## Gold Nanoparticles

(leer)

## Other Reporter System

(leer)

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# 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:

1. Excess TCEP relative to ssDNA (standard method)
2. Excess ssDNA relative to TCEP (literature-based method)
3. 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.

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# Material and Methods

Developing a rapid and filed-ready test:
The CRISPR Cas12a detecting Arsenophonus

## Production and Purification

Glycerol Stock Preparation of Cas12a Plasmids in E. coli
Glycerol stocks of Cas12a plasmids were prepared in Escherichia coli. The plasmids 6His-MBP-TEV-huAsCas12a (pIGEM25\_001, Amp^R, Addgene; Addgene plasmid #90095), pMBP-FnCas12a (pIGEM25\_002, Amp^R, Addgene; Addgene plasmid #113432) and 6His-MBP-TEV-huLbCas12a (pIGEM25\_003, Amp^R, Addgene, Watertown, MA, USA; Addgene plasmid #90096) were received from Addgene and initially stored at 4 °C. For transformation, LB agar (X969.2, Carl Roth, Karlsruhe, Germany) was supplemented with carbenicillin disodium (CAS 4800-94-6, BIC0109, Apollo Scientific, Manchester, UK) to a final concentration of 100 µg/mL and poured into plates under sterile conditions. Dilution smears were prepared for each plasmid, and plates were incubated overnight at 37 °C. Single colonies obtained from these plates were inoculated into 10 mL LB medium (X968.4, Carl Roth, Karlsruhe, Germany) supplemented with carbenicillin disodium (100 µg/mL). For each plasmid, three independent overnight cultures were prepared, resulting in nine cultures in total, in addition to a negative control. Cultures were incubated overnight at 37 °C and 180 rpm.
Optical densities (OD) were determined by measuring diluted culture samples (1:10) at 600 nm using a spectrophotometer (XXXXX, XXXXX, XXXXX). Two glycerol stocks were prepared from each culture by mixing 500 µL cell suspension with 500 µL glycerol (XXXXX, XXXXX, XXXXX) in cryotubes, which were subsequently stored at −80 °C.
Plasmid DNA was extracted from 600 µL of culture using the PureYield Plasmid Miniprep System (Promega, Madison, WI, USA), and DNA was eluted in 30 µL elution buffer following 5 min incubation at room temperature. DNA concentrations and purity were assessed with a NanoDrop spectrophotometer (XXXXX, XXXXX, XXXXX). For quality control, 0.8% agarose gel electrophoresis was performed. Samples were mixed with ddH₂O and 6X loading dye (XXXXX, XXXXX, XXXXX) according to the calculated DNA amounts and separated at 150 V, 500 mA, and 120 W for 30 min. A 1 kb Plus DNA ladder (XXXXX, XXXXX, XXXXX) was used as molecular weight reference. Selected plasmid preparations were subsequently sent for sequencing (Microsynth AG, Balgach, Switzerland) to confirm identity.

Preparation of chemically competent cells for Protein expression
Chemically competent E. coli Rosetta (DE3) cells (Novagen, Sigma-Aldrich, St. Louis, MO, USA; product no. 70954) were prepared for protein expression. For preculture, 10 mL LB medium (X968.4, Carl Roth, Karlsruhe, Germany) were inoculated with Rosetta (DE3) cells and incubated overnight at 37 °C and 180 rpm in a 50 mL shake flask. The following day, 100 mL LB medium were inoculated with 1 mL of the overnight culture and incubated at 37 °C and 230 rpm until an OD₆₀₀ of 0.5 – 0.6 was reached, corresponding to the mid-logarithmic growth phase.
From this culture, 35 mL were transferred into a 50 mL centrifuge tube and placed on ice for 20 min. Cells were pelleted by centrifugation at 3900 rpm and 4 °C for 5 min in a precooled centrifuge (XXXXX, XXXXX, XXXXX), and the supernatant was discarded. The pellet was resuspended in 20 mL of ice-cold 100 mM calcium chloride (CAS 10035-04-8, 423525000, Acros Organics, Geel, Belgium) and incubated on ice for 1 h. Cells were again centrifuged at 4500 rpm and 4 °C for 5 min, and the supernatant was discarded. The resulting pellet was gently resuspended in 2 mL of ice-cold 100 mM calcium chloride containing 15 % (v/v) glycerol (CAS 56-81-5, 7530.5, Carl Roth, Karlsruhe, Germany). Aliquots of 100 µL were prepared in sterile 1.5 mL tubes on ice and immediately stored at −80 °C.

Heterologous Expression of CRISPR-Cas12a/Cas12a (As/Fn/Lb)
Heterologous expression of CRISPR-Cas12a/Cas12a variants was performed in E. coli Rosetta (DE3) competent cells (prepared as described in “Preparation of chemically competent cells for protein expression”). The plasmids 6His-MBP-TEV-huLbCas12a (pIGEM25\_003, Amp^R, Addgene, Watertown, MA, USA; Addgene plasmid #90096), 6His-MBP-TEV-huAsCas12a (pIGEM25\_001, Amp^R, Addgene; Addgene plasmid #90095) and pMBP-FnCas12a (pIGEM25\_002, Amp^R, Addgene; Addgene plasmid #113432) were used. Transformation was carried out using 1 µL plasmid DNA, and transformed cells were streaked onto LB agar plates (X969.2, Carl Roth, Karlsruhe, Germany) containing carbenicillin disodium (CAS 4800-94-6, BIC0109, Apollo Scientific, Manchester, UK) at a final concentration of 100 µg/mL. Plates were incubated overnight at 37 °C, and single colonies were selected for preculture.
For preculture, 20 mL LB medium (X968.4, Carl Roth, Karlsruhe, Germany) supplemented with carbenicillin disodium (100 µg/mL) were inoculated with single colonies and incubated at 37 °C and 160 rpm overnight. The following day, 15 mL of each preculture were transferred into 1 L Terrific Broth medium (XXXXX, XXXXX, XXXXX) containing carbenicillin disodium (100 µg/mL) and incubated at 37 °C and 150 rpm. During exponential growth, aliquots of 500 µL culture were mixed with 500 µL 50 % (v/v) glycerol (CAS 56-81-5, 7530.5, Carl Roth, Karlsruhe, Germany) and stored at −80 °C as glycerol stocks.
At an OD₆₀₀ of approximately 0.5, cultures were placed on ice for 15 min, and protein expression was induced by adding 1 M IPTG (CAS 367-93-1, XXXXX, XXXXX) to a final concentration of 0.133 mM. Cultures were incubated overnight at 18 °C and 120 rpm for approximately 16 h. After induction, 1 mL culture aliquots were centrifuged, and the pellets were stored at −20 °C. Main cultures were harvested by centrifugation at 3214 g and 4 °C for 30 min, and cell pellets were collected and stored in 50 mL centrifuge tubes at −20 °C.
For expression analysis, frozen pellets were resuspended in 100 µL deionized water, mixed with sample buffer (XXXXX, XXXXX, XXXXX), and subjected to SDS-PAGE on 15% polyacrylamide gels. Electrophoresis was performed at 200 V and 50 mA for 50 min. Gels were stained with QuickBlue Protein Stain (XXXXX, XXXXX, XXXXX) for 2 h and subsequently incubated in water until documentation.

Cas12a Purification
Cell pellets containing heterologously expressed Cas12a in E. coli Rosetta (DE3) were thawed on ice. The pellets were resuspended in lysis buffer (500 mM NaCl, 20 mM Tris, 10 mM imidazole, pH 8.0) at a ratio of 1 g pellet : 4 mL buffer. To the suspension, lysozyme was added (10 µL per mL buffer) together with 0.5–1% Triton X-100. After incubation on ice, the lysate was supplemented with MgCl₂ (final 1 mM) and supernuclease (25 U/mL lysate) and further incubated on ice with occasional mixing.
For cell disruption, the lysate was subjected to ultrasonication (3 cycles of 5 min sonication at 80% amplitude with 5 min cooling on ice between cycles). Cell debris was removed by ultracentrifugation at 30’000 g, 4 °C, 45 min, yielding a clarified supernatant. The supernatant was filtered through a 0.2 µm filter prior to affinity purification.
Purification was performed via immobilized metal affinity chromatography (IMAC, HisTrap HP column, Cytiva). The column was equilibrated with 5 column volumes (CV) lysis buffer, and the clarified lysate was loaded. After binding, the column was washed with 5 CV of wash buffer (500 mM NaCl, 20 mM Tris, 20 mM imidazole, pH 8.0) to remove nonspecifically bound proteins. Cas12a was eluted with elution buffer (500 mM NaCl, 20 mM Tris, 250 mM imidazole, pH 8.0). Eluate fractions corresponding to the Cas12a peak were pooled.
For buffer exchange and formulation, the pooled eluates were applied to desalting columns (PD-10, Cytiva) equilibrated with formulation buffer (20 mM HEPES pH 7.5, 150 mM KCl, 5% glycerol, 0.5 mM DTT). Typically, 1 volume sample was applied, and 1.4 volumes formulation buffer were used for elution. The desalted protein was collected, aliquoted and stored at –80 °C.
Protein concentration was routinely monitored via Nanodrop (A₂₈₀, ε = XX M⁻¹ cm⁻¹ for Cas12a) across key steps (lysate, wash, elution, final formulation). Typical yields were in the range of 10–15 mg/mL after formulation.
Protein integrity and purity were analyzed by SDS–PAGE (12% Tris–Glycine gels). Samples were diluted in formulation buffer, mixed with loading dye, heat-denatured (95 °C, 5 min), and loaded (10 µL per lane). Gels were run at 200 V for 50 min and stained with QuickBlue. Prominent Cas12a bands at the expected molecular weight (~150 kDa) were observed, with negligible contaminant proteins after IMAC and buffer exchange.

## Base Test Fluorescence Reporter

Base Test Fluorescence Reporter
Fluorescence reporter assays based on Cas12a 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 labeled with a fluorophore and quencher was used to monitor cleavage activity (Cas12 ssDNA Reporter, Signalchem Diagnotstics, 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 labeled with a fluorophore and quencher. 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 adjusted to pH 8.0. Fluorescence was recorded in a FlexStation 3 plate reader (Molecular Devices) at 485 nm excitation and 520 nm emission. Kinetic measurements were performed at 25 °C or 30 °C with 5 min-intervals for up to 16 h, or as endpoint assays at specified times. Negative controls lacking Cas12a, crRNA, or target DNA were included in each experiment.

Buffer pH Comparison
An in-house 10X Cas12 reaction buffer (1 M NaCl, 50 mM MgCl₂·6H₂O, 200 mM HEPES, 1 mM EDTA in nuclease-free water) was prepared and adjusted either to pH 6.5 or pH 8.0 prior to dilution. Initial assays were performed with both buffer variants to assess the effect of pH on nuclease activity. Cas12a variants were tested in duplicate under these conditions.

Magnesium Chloride Dependency
To assess ion dependency, reactions were carried out in modified buffers lacking Mg²⁺. MgCl₂ was subsequently added in defined concentrations by spiking from a stock solution, while in parallel reactions equimolar EDTA was included to bind Mg²⁺. Different Mg²⁺/EDTA ratios were tested at 30 °C reaction temperature to determine the optimal free magnesium concentration required for Cas12a activity.

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
Genomic DNA was extracted from Pseudomonas putida 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, crRNA\_iGEM25\_007 and crRNA\_iGEM25\_008, 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 Arseph infection (provided by BIOREBA, Rheinach BL, Switzerland) were tested as additional targets in the Cas12a fluorescence reporter assay. crRNAs were designed against annotated Arseph 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.

## Gold Nanoparticles

## Other Reporter Systems

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