In 2017, Bio Bureau started a research program for the biotechnological control of the golden mussel, funded by China Tree Gorges – CTG Brasil, through ANEEL’s research and development program.
We divided the development into stages according to the Technology Readiness Scale – TRL. This division facilitates the understanding and communication of a project with a high degree of complexity.
| Technology Readiness Level | Technological goals |
| TRL #1 Basic Principles | - Open field performance of Aedes aegypti mosquitoes designed to control dengue and other diseases (Harris et al., 2011)
- Scientific evidence that genetically modified organisms can limit the population growth of mosquito malaria vectors in laboratory conditions (Windbichler et al., 2011; Hammond et al., 2016)
- The genome of the golden mussel is published (Uliano da Silva et al., 2018)
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| TRL #2 Conceptual model | - In this article, we conceptualize a GM mussel with self-limiting demographic characteristics based on the sexual distortion of the CRISPR-Cas9 gene: GM females are infertile while GM males spread female infertility in the population
- Technical and economic feasibility and sustainability studies are carried out.
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| TRL #3 Proof of concept | - Identification of haplosufficient somatic female fertility genes in the genome of Limnoperna fortunei
- Computer simulations of the dynamics of homing endonuclease genes (HEG) together with musseldemo
- Construction of CRISPR-Cas9 gene delivery vectors targeting female fertility genes
- Genetic transformation of mussel cells.
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| TRL #4 Lab-scale prototype | - Generation of genetically modified mussels with the CRISPR-Cas9 allele
- Genotyping, phenotyping and demonstration of haplosufficiency
- Laboratory testing of GM mussels in experimental aquariums under highly controlled conditions according to phase 1 of the World Health Organization (WHO) guidelines
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| TRL #5 Field-scale prototype | - Breeding GM mussels with wild-type specimens in controlled mesocosm experiments
- Empirical evaluation of homing rate and CRISPR homing allele frequency in 4-generation experiments (phase 2 of WHO guidelines)
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| TRL # 6 Scaling up | - Production of large (10^9) quantities of GM embryos that can withstand transportation for several hours (12-24h) to be seeded in laboratories by a reservoir
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| TRL #7 Pilot test | - Open field performance of GM mussels (phase 3 of WHO guidelines)
- Disinfestation of a reservoir branch upstream of a power plant using GM mussels in cages, in conjunction with chemical disinfestation of the plant
- Monitoring reinfestation and transgenic DNA using quantitative PCR (phase 4 of the WHO guidelines) .
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| TRL #8 Regulatory stage | - Evaluation of the biosafety and efficacy of the genetic elements introduced
- Standard Operating Procedures for producing GM embryos, GM adults, planting seeds, GM mussels in cages, etc.
- Licenses, certificates and permits from MAPA (Ministry of Agriculture, Livestock and Supply), IBAMA, CTNBio (Brazilian Biosafety Technical Commission)
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| TRL #9 Go to the market | - Successful disinfestation of entire hydroelectric reservoir
- Disinfestation as a service available on the market.
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The description of the stages was formalized in an article published in pre-print format in 2018.
- Rebelo MF, Afonso LF, Americo JA, da Silva L, Neto JLB, Dondero F, Zhang Q. 2018. A sustainable synthetic biology approach for the control of the invasive golden mussel (Limnoperna fortunei) PeerJ Preprints6:e27164v3 https://doi.org/10.7287/peerj.preprints.27164v3
