Production of Cas12a

Results

This section presents the results of the in-house production of Cas12a.

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Figure 1: Graphical abstract of production workflow. A plasmid encoding the Cas12a gene was transformed into E. coli Rosetta (DE3) cells (1). A single colony was selected (2), and protein expression was induced with IPTG (3). The cells were lysed (4), and the soluble fraction was purified by immobilized metal affinity chromatography (IMAC) (5), followed by buffer exchange into the final formulation buffer (6). Protein quality and functionality were assessed by SDS-PAGE (7) and an activity assay (8). Created with Biorender..

In total, we expressed three Cas12a variants (FnCas12a, AsCas12a, and LbCas12a). The harvested biomass varied between variants (Figure 2 A), but yields were comparable to, or in some cases higher than, those reported in the literature [1]. Due to restricted lab access, only AsCas12a and LbCas12a were purified. Nevertheless, the obtained protein amounts were sufficient to support our iGEM experiments. Final yields were 43.03 mg protein per g dry pellet (118.8 mg protein per L culture) for AsCas12a, and 28.19 mg protein per g dry pellet (151.1 mg protein per L culture) for LbCas12a (Figure 2 B and C). SDS-PAGE confirmed the successful expression and purification of Cas12a, with strong bands observed at the expected size of ~150-160 kDa (Figure 3).

A beautiful sunset over the mountains
Figure 2: Yields of Cas12a production. (A) Harvested biomass for each variant. (B) Final protein yield normalized to biomass. (C) Final protein yield normalized to culture volume.
A beautiful sunset over the mountains
Figure 3: SDS-PAGE of in-house produced Cas12a by purification step. Lanes 2 to 6 represents the purification of AsCas12a, while lanes 7 to 11 represents the LbCas12a.

Although additional polishing steps (His-tag cleavage, further chromatography, and buffer exchange) were initially planned, the proteins already displayed activity in downstream assays. Therefore, purification was stopped at this stage. The His-tag may also serve as an immobilization handle for future applications such as rapid flow tests.

For activity comparison, we also purchased commercial LbCas12a (see “Test of Cas12a”). Since we performed only single expression and purification runs, the yields between variants should be considered preliminary and not directly comparable.

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Figure 4: Cost comparison. Estimated costs of a PCR test [2] versus Cas12a required for one test. Please note the logarithmic y-axis.

To assess the value of in-house production, we performed a rough cost estimation considering materials, consumables, and labor. Our analysis shows that in-house production can reduce costs by up to 99.8% compared to purchasing commercial Cas12a. Although purity is lower, the proteins remained fully functional in our test systems (see “Test of Cas12a ”). Based on our yields and the estimated requirement of 0.4 µg per reaction, one batch of in-house Cas12a is sufficient for approximately 75’000 tests. This corresponds to ~0.013 CHF per test, compared to ~5.30 CHF per test with commercially purchased protein. Thus, in-house production provides Cas12a protein at sufficient quality for truly low-cost diagnostic testing.

Material and Methods

The purification process was carried out as outlined in Figure 1. The overall expression and purification workflow was adapted from Mohanraju et al. [1].

Glycerol Stock Preparation of Cas12a Plasmids in E. coli Rosetta (DE3)

Cas12a plasmids were obtained from Addgene (Addgene, Watertown, MA, USA):

  • 6His-MBP-TEV-huAsCas12a (pIGEM25_001, AmpR; Addgene #90095)
  • pMBP-FnCas12a (pIGEM25_002, AmpR; Addgene #113432)
  • 6His-MBP-TEV-huLbCas12a (pIGEM25_003, AmpR; Addgene #90096)

Plasmids were received and stored at 4 °C until use. For transformation, LB agar (X969.2, Carl Roth, Karlsruhe, Germany) was supplemented with carbenicillin (100 µg/mL; CAS 4800-94-6, BIC0109, Apollo Scientific, Manchester, UK). Dilution streaks were prepared, plates were incubated overnight at 37 °C, and single colonies were selected. Colonies were grown in 10 mL LB medium with carbenicillin (100 µg/mL) overnight at 37 °C and 180 rpm. Three independent overnight cultures per plasmid were prepared, along with a negative control.

Two glycerol stocks were prepared from each culture (500 µL culture + 500 µL sterile glycerol; Carl Roth, Karlsruhe, Germany) and stored at −80 °C. Plasmid DNA was isolated using the PureYield Miniprep System (Promega) and quantified by NanoDrop spectrophotometry. Plasmid integrity was verified by 0.8% agarose gel electrophoresis, and selected samples were confirmed by sequencing (Microsynth AG, Balgach, Switzerland).

Preparation of Chemically Competent E. coli Rosetta (DE3)

Chemically competent E. coli Rosetta (DE3) cells (Novagen, Sigma-Aldrich, St. Louis, MO, USA) were prepared by CaCl₂ treatment. Briefly, cultures were grown in LB medium (X968.4, Carl Roth, Karlsruhe, Germany ) to mid-log phase (OD₆₀₀ ≈ 0.5–0.6), chilled on ice, pelleted by centrifugation, and resuspended in ice-cold 100 mM CaCl₂. After incubation and re-pelleting, cells were resuspended in 100 mM CaCl₂ with 15% glycerol, aliquoted, and stored at −80 °C.

Heterologous Expression of Cas12a variants

Chemically competent Rosetta (DE3) cells were transformed with 1 µL Cas12a plasmids and plated on LB agar containing carbenicillin (100 µg/mL). After overnight incubation, single colonies were inoculated into LB medium with carbenicillin for preculture.
Precultures were transferred into Terrific Broth (TB) medium containing carbenicillin and grown at 37 °C until OD₆₀₀ ≈ 0.5. Protein expression was induced with IPTG (final concentration 0.133 mM) and cultures were incubated overnight at 18 °C, 120 rpm. Cells were harvested by centrifugation (3214 g, 4 °C, 30 min) and stored at −20 °C.

Cas12a purification

Cell pellets were thawed on ice and resuspended in lysis buffer (500 mM NaCl, 20 mM Tris, 10 mM imidazole, pH 8.0). Lysozyme, Triton X-100 (0.5–1%), MgCl₂ (1 mM), and supernuclease (25 U/mL) were added. Cells were disrupted by ultrasonication (3 cycles, 5 min, 80% amplitude, cooling on ice between cycles). Debris was removed by centrifugation (30’000 g, 4 °C, 45 min), and the supernatant was filtered (0.2 µm).
Purification was performed using immobilized metal affinity chromatography (IMAC, HisTrap HP column, Cytiva). After column equilibration, lysate was loaded, washed with buffer (500 mM NaCl, 20 mM Tris, 20 mM imidazole, pH 8.0), and eluted with buffer containing 250 mM imidazole. Fractions containing Cas12a were pooled and desalted (PD-10, Cytiva) into formulation buffer (20 mM HEPES pH 7.5, 150 mM KCl, 5% glycerol, 0.5 mM DTT).
Protein concentration was determined by NanoDrop at 280 nm, and purity was assessed by SDS-PAGE. Prominent bands confirmed the presence of Cas12a at the expected molecular weight (~150–160 kDa).

Notebooks

In this section, the Notebooks are uploaded.

References

[1] P. Mohanraju, J. Van Der Oost, M. Jinek, und D. C. Swarts, „Heterologous Expression and Purification of CRISPR-Cas12a/Cpf1“, Bio Protoc, Bd. 8, Nr. 9, S. e2842, Mai 2018, doi: 10.21769/BioProtoc.2842. [2] N. Minhas u. a., „Cost-analysis of real time RT-PCR test performed for COVID-19 diagnosis at India’s national reference laboratory during the early stages of pandemic mitigation“, PLOS ONE, Bd. 18, Nr. 1, S. e0277867, Jan. 2023, doi: 10.1371/journal.pone.0277867.