Periodic Reporting for period 1 - CRISPART (A highly efficient CRISPR/Cas9 gene editing method for difficult to transform arthropods)
Periodo di rendicontazione: 2023-09-01 al 2025-02-28
Controlling arthropods is crucial for crop protection and human and animal health. Biotechnological advances are needed to develop safer pest management products. Gene editing using CRISPR/Cas9 has revolutionized genetics, typically by injecting the Cas9 ribonucleoprotein (RNP) complex into embryos. However, in Chelicerata — a large group including ticks, mites, and spiders — embryo injection has proven nearly impossible. For mites like Varroa destructor, Dermatophagoides pteronyssinus, and Tetranychus urticae, embryo injection consistently fails, despite intensive efforts. Similar challenges exist for many insect pests, mainly due to difficulties in collecting eggs rather than technical barriers. Thus, there is a need for a gene-editing method that bypasses embryo injection.
An alternative is injecting RNPs into adult females near the ovaries, a method tested in nematodes, crustaceans, and some insects. Using this approach, our group achieved the first stable genetic transformation of a chelicerate, T. urticae, in 2020, proving CRISPR/Cas9 editing possible in this group. However, transformation efficiency remained low (<0.5%), limiting its practical use.
To overcome this, we developed SYNCAS, a method combining Branched Amphipathic Peptide Capsules (BAPC) and Quillaja saponin with Cas9 RNPs. This synergy boosts delivery to oocytes, raising editing efficiencies above 10%. SYNCAS enabled successful editing not only of T. urticae but also of the thrips Frankliniella occidentalis, a species with embryos difficult to collect and inject.
This breakthrough offers a transformative tool for genetic modification of previously intractable arthropods, with major implications for pest control, agricultural biotechnology, and genetic research. High-efficiency knock-ins and knock-outs could reduce dependence on pesticides, aligning with the EU’s goal to cut pesticide use by 50% by 2030. The method’s commercial potential is significant, given the current limitation of gene-editing services to model organisms like Drosophila.
Building on successes in T. urticae and F. occidentalis, the project aimed to extend SYNCAS to other economically important species where traditional methods fail. Four candidates were selected: (1) Amblyseius swirskii, a predatory mite vital to biocontrol but never genetically transformed; (2) Bemisia tabaci, the silverleaf whitefly, difficult to inject; (3) Myzus persicae, an aphid with a complex, clonal life cycle; and (4) Nezara viridula, the southern green stinkbug, intractable due to injection challenges. Success in these species would mark major advances.
We also investigated SYNCAS's mode of action. Although BAPCs aid nucleic acid uptake and saponins enhance membrane permeability, only their combination dramatically boosts editing efficiency. We explored whether this synergism occurs pre- or post-injection by varying timing and testing alternative formulations.
To ensure long-term impact, we developed an intellectual property strategy, conducted a patent landscape analysis, and engaged with agrochemical companies to assess market interest. Interviews and market analyses informed the potential creation of a spin-off company focused on arthropod gene editing.
Through this integrated approach, we aimed to establish a broadly applicable gene-editing tool and position it for widespread research and industrial use, paving the way for advances in pest control, biotechnology, and fundamental biology.
An alternative is injecting RNPs into adult females near the ovaries, a method tested in nematodes, crustaceans, and some insects. Using this approach, our group achieved the first stable genetic transformation of a chelicerate, T. urticae, in 2020, proving CRISPR/Cas9 editing possible in this group. However, transformation efficiency remained low (<0.5%), limiting its practical use.
To overcome this, we developed SYNCAS, a method combining Branched Amphipathic Peptide Capsules (BAPC) and Quillaja saponin with Cas9 RNPs. This synergy boosts delivery to oocytes, raising editing efficiencies above 10%. SYNCAS enabled successful editing not only of T. urticae but also of the thrips Frankliniella occidentalis, a species with embryos difficult to collect and inject.
This breakthrough offers a transformative tool for genetic modification of previously intractable arthropods, with major implications for pest control, agricultural biotechnology, and genetic research. High-efficiency knock-ins and knock-outs could reduce dependence on pesticides, aligning with the EU’s goal to cut pesticide use by 50% by 2030. The method’s commercial potential is significant, given the current limitation of gene-editing services to model organisms like Drosophila.
Building on successes in T. urticae and F. occidentalis, the project aimed to extend SYNCAS to other economically important species where traditional methods fail. Four candidates were selected: (1) Amblyseius swirskii, a predatory mite vital to biocontrol but never genetically transformed; (2) Bemisia tabaci, the silverleaf whitefly, difficult to inject; (3) Myzus persicae, an aphid with a complex, clonal life cycle; and (4) Nezara viridula, the southern green stinkbug, intractable due to injection challenges. Success in these species would mark major advances.
We also investigated SYNCAS's mode of action. Although BAPCs aid nucleic acid uptake and saponins enhance membrane permeability, only their combination dramatically boosts editing efficiency. We explored whether this synergism occurs pre- or post-injection by varying timing and testing alternative formulations.
To ensure long-term impact, we developed an intellectual property strategy, conducted a patent landscape analysis, and engaged with agrochemical companies to assess market interest. Interviews and market analyses informed the potential creation of a spin-off company focused on arthropod gene editing.
Through this integrated approach, we aimed to establish a broadly applicable gene-editing tool and position it for widespread research and industrial use, paving the way for advances in pest control, biotechnology, and fundamental biology.
WP1: Demonstration of Method Efficacy on Economically Relevant Arthropods
Goal: Extend the SYNCAS method (combining BAPC, Quillaja saponin, and CRISPR/Cas9) beyond Tetranychus urticae and Frankliniella occidentalis to other difficult-to-transform arthropods. Focus was on achieving knock-outs in Amblyseius swirskii, Bemisia tabaci, Myzus persicae, and Nezara viridula.
Results:
Successful knock-outs in three of four target species.
A. swirskii and N. viridula: Up to ~5% of progeny carried KO mutations.
B. tabaci: Editing efficiency reached ~40%.
M. persicae: No gene editing detected, suggesting SYNCAS is less effective for viviparous species.
Additional species tested: Phytoseiulus persimilis (predatory mite), with similar results as A. swirskii.
Results published in Insect Biochemistry and Molecular Physiology (https://doi.org/10.1016/j.ibmb.2024.104232(si apre in una nuova finestra)).
WP2: Investigating the Mechanism Behind SYNCAS Synergy
Goal: Understand why the combination of BAPC and Quillaja saponin enables efficient transformation, whereas each component alone does not.
Results:
Sequential Injection Experiments: When BAPC and saponin were injected separately, editing efficiency dropped from 10–20% to 1.5–2%, suggesting that a physical interaction is critical.
Testing Other Endosomal Escape Agents: Compounds like Tween-20, Triton-X, CHAPS, and PEI failed to replicate the SYNCAS effect, ruling out a simple additive effect.
Alternative Saponins: Testing sasanqua saponin and Saikosaponin D showed no replacement of Quillaja saponin, suggesting a specific chemical requirement.
Conclusion: The synergistic effect likely depends on a specific interaction between BAPC and components within the heterogeneous Quillaja saponin mixture. Further work is needed to identify the exact active molecule(s).
WP3: Intellectual Property (IPR) Strategy and Market Interest
Goal: Secure intellectual property and evaluate commercialization options.
Results:
Patent application PCT/EP2024/069484 filed in July 2023, strengthened with new experimental data from A. swirskii and B. tabaci.
Discussions held with major agrochemical companies (BASF, Bayer, Syngenta, ISK, Nichino) showed strong interest in using SYNCAS, but companies preferred service agreements over licensing.
Thomas Van Leeuwen’s lab is currently offering SYNCAS-based services to these companies.
A spin-off company is being explored to offer independent services for investigating the mode of action (MoA) of new insecticide leads, with a business model and financial plan under development.
Goal: Extend the SYNCAS method (combining BAPC, Quillaja saponin, and CRISPR/Cas9) beyond Tetranychus urticae and Frankliniella occidentalis to other difficult-to-transform arthropods. Focus was on achieving knock-outs in Amblyseius swirskii, Bemisia tabaci, Myzus persicae, and Nezara viridula.
Results:
Successful knock-outs in three of four target species.
A. swirskii and N. viridula: Up to ~5% of progeny carried KO mutations.
B. tabaci: Editing efficiency reached ~40%.
M. persicae: No gene editing detected, suggesting SYNCAS is less effective for viviparous species.
Additional species tested: Phytoseiulus persimilis (predatory mite), with similar results as A. swirskii.
Results published in Insect Biochemistry and Molecular Physiology (https://doi.org/10.1016/j.ibmb.2024.104232(si apre in una nuova finestra)).
WP2: Investigating the Mechanism Behind SYNCAS Synergy
Goal: Understand why the combination of BAPC and Quillaja saponin enables efficient transformation, whereas each component alone does not.
Results:
Sequential Injection Experiments: When BAPC and saponin were injected separately, editing efficiency dropped from 10–20% to 1.5–2%, suggesting that a physical interaction is critical.
Testing Other Endosomal Escape Agents: Compounds like Tween-20, Triton-X, CHAPS, and PEI failed to replicate the SYNCAS effect, ruling out a simple additive effect.
Alternative Saponins: Testing sasanqua saponin and Saikosaponin D showed no replacement of Quillaja saponin, suggesting a specific chemical requirement.
Conclusion: The synergistic effect likely depends on a specific interaction between BAPC and components within the heterogeneous Quillaja saponin mixture. Further work is needed to identify the exact active molecule(s).
WP3: Intellectual Property (IPR) Strategy and Market Interest
Goal: Secure intellectual property and evaluate commercialization options.
Results:
Patent application PCT/EP2024/069484 filed in July 2023, strengthened with new experimental data from A. swirskii and B. tabaci.
Discussions held with major agrochemical companies (BASF, Bayer, Syngenta, ISK, Nichino) showed strong interest in using SYNCAS, but companies preferred service agreements over licensing.
Thomas Van Leeuwen’s lab is currently offering SYNCAS-based services to these companies.
A spin-off company is being explored to offer independent services for investigating the mode of action (MoA) of new insecticide leads, with a business model and financial plan under development.
This project proved proof of concept for the broad applicability of the SYNCAS method in hard to transform arthropods which will greatly progress arthropod research for academia and industry. Potential additional research could involve the demonstration of a "knock-in" protocol where specifically designed (desired) mutations are introduced genes of these arthropods as this can further strengthen the valorization potential of the SYNCAS method. The results of this ERC PoC will help industry to develop more sustainable insecticides and acaricides. The SYNCAS technology allows companies to understand the mode of action of the selected leads and may allow them to anticipate to existing resistance in certain insect species. The SYNCAS technology will be offered worldwide to agrochemical companies.
In addition, after some years of experience with the SYNCAS technology and the many services for companies, Professor Thomas Van Leeuwen may operate as expert to provide support in European policy making on Integrated Pest Management
In addition, after some years of experience with the SYNCAS technology and the many services for companies, Professor Thomas Van Leeuwen may operate as expert to provide support in European policy making on Integrated Pest Management