CRISPR piwil1 Gene Knockout for Sterile Salmon Production
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Solution Overview
Problem
The salmon aquaculture industry faces challenges such as genetic introgression from escaped farmed salmon into wild populations, aggressive and sexual behavior issues, and suboptimal growth and meat quality due to the lack of effective methods to inhibit sexual activity in fish, with existing methods like triploidization being costly, labor-intensive, and affecting fish welfare.
Innovation Solution
A method involving the modification of fish zygotes or embryos to eliminate functional expression of the piwil gene, which is essential for germ cell survival, allowing for the production of sterile offspring while maintaining fertile broodstock capable of producing viable gametes, thereby preventing interbreeding with wild fish.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If triploidisation is used to produce sterile salmon, then sterility is achieved, but fish welfare deteriorates due to vertebral deformities and cataracts
Solution Approach 1:
The invention extracts and eliminates the harmful triploid chromosome condition while retaining the desired sterility outcome. By using CRISPR/Cas9 to specifically target and disable germ cell development through piwil1 gene knockout, the method removes the need for triploidisation and its associated welfare problems while achieving complete sterility.
Solution Approach 2:
The invention replaces the mechanical/chromosomal manipulation of triploidisation with a molecular biology approach using CRISPR/Cas9 gene editing. This substitution allows for precise genetic modification at the molecular level rather than relying on chromosomal abnormalities, thereby achieving sterility without the physical deformities associated with triploidisation.
2Reliability
If triploidisation is used to produce sterile salmon, then sterility is achieved, but production completeness deteriorates with 5-20% diploid fish remaining
Solution Approach 1:
The invention replaces the imprecise mechanical process of triploidisation with the precise molecular targeting of CRISPR/Cas9 gene editing. The piwil1 gene knockout ensures complete and uniform sterility across all fish, eliminating the 5-20% diploid contamination that occurs with triploidisation.
Solution Approach 2:
The invention changes the fundamental parameter from chromosomal number manipulation to specific gene function elimination. By targeting the piwil1 gene essential for germ cell development, the method achieves consistent 100% sterility regardless of ploidy level, eliminating the variability inherent in triploidisation.
3Reliability
If surgical castration is used in mammals, then reproduction is inhibited, but the method is not applicable to fish due to internal testis location and large animal numbers
Solution Approach 1:
The invention replaces the mechanical surgical approach with a molecular biology approach using CRISPR/Cas9 gene editing and mRNA injection. This substitution enables non-invasive sterility induction that can be applied to fish embryos regardless of testis location or animal size, making the method scalable to large fish populations.
Solution Approach 2:
The invention performs the sterility-inducing action preliminarily during the embryonic stage through mRNA or protein injection, before the testis develops and becomes internally located. This preliminary genetic modification ensures sterility is established early in development, avoiding the need for later surgical intervention.
4Duration of action of moving object
If hormone vaccination is used for short term castration, then puberty is temporarily delayed, but reproduction is not inhibited
Solution Approach 1:
The invention extracts and eliminates the temporary nature of hormone vaccination by permanently disabling germ cell development through CRISPR/Cas9-mediated piwil1 gene knockout. This permanent genetic modification ensures complete and lasting sterility rather than temporary puberty delay.
Solution Approach 2:
The invention replaces the temporary hormonal suppression mechanism with a permanent molecular genetic modification. By using CRISPR/Cas9 to create heritable piwil1 gene knockouts, the method achieves permanent sterility that is passed to subsequent generations, eliminating the need for repeated vaccinations.
5Reliability
If vivo-morpholinos are used to block germ cell mRNA in zebrafish, then germ cell development is blocked, but the method is laborious and expensive for treating every egg batch
Solution Approach 1:
The invention extracts and eliminates the need for treating every individual egg batch by creating a heritable genetic modification. The CRISPR/Cas9-mediated piwil1 knockout is established in the germline, so all subsequent offspring inherit the sterile trait automatically, eliminating the need for repeated treatments.
Solution Approach 2:
The invention performs the germ cell blocking action preliminarily by establishing the piwil1 knockout in the founding broodstock. This preliminary genetic modification is then heritably transmitted to all subsequent generations, achieving continuous sterility without the need for ongoing treatment of each egg batch.
6Reliability
If vivo-morpholinos are used to block germ cell mRNA, then sterility is induced, but the solution may be toxic to embryos and not 100% effective
Solution Approach 1:
The invention replaces the toxic chemical morpholino approach with a precise molecular genetic editing approach using CRISPR/Cas9. This substitution eliminates the embryo toxicity associated with morpholinos while achieving complete and heritable sterility through targeted piwil1 gene disruption.
Solution Approach 2:
The invention extracts and eliminates the toxic effects of morpholino solutions by using CRISPR/Cas9 gene editing instead. The genetic modification approach achieves the same germ cell blocking effect without the chemical toxicity that harms embryonic development.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach ensures 100% sterile offspring, preserving genetic integrity and improving fish welfare by preventing genetic introgression and reducing aggressive behavior, while allowing for sustainable genetic trait addition.
Implementation Method 1
modifying the genome of a fish zygote or the genome of one or more cells of an early-stage fish embryo to eliminate functional expression of a germ cell survival factor gene, wherein the germ cell survival factor gene is a piwil gene or a piwi gene
Implementation Method 2
introducing a CRISPR enzyme (e.g. Cas9) and/or a gRNA comprising a piwil or piwi gene-targeting sequence
Data Source
AI summary
The present invention relates, inter alia, to processes for making modified fish zygotes or early-stage fish embryos (particularly salmon zygotes and salmon embryos). The invention also provides fish zygotes, fish embryos, juvenile fish, mature fish and sterile fish which are produced by the processes of the invention.