GENERATION OF KNOCK-IN TRANSGENIC CHANNEL CATFISH Ictalurus punctatus CARRYING ELONGASE GENE FROM MASU SALMON Oncorhynchus masou AND DESATURASE GENES FROM RABBITFISH Siganus canaliculatus USING CRISPR/CAS9, TOL2 TRANSPOSON SYSTEM AND DOUBLE ELECTROPORATION APPROACHES
Channel catfish (Ictalurus punctatus) is a very popular freshwater food fish consumed in the USA, but unlike marine fish, it contains low levels of n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic acid (EPA;20:5n-3) and docosahexaenoic acid(DHA;22:6n-3). EPA and DHA have important beneficial effects on human health, such as prevention of cardiovascular disease. To modify the HUFA metabolic pathway to produce transgenic channel catfish lines with high levels of n-3 HUFA, one elongase transgene (Elovl2 isolated from masu salmon) and two desaturase transgenes (Δ4 and Δ5/6 desaturase genes isolated from Rabbitfish) driven by the carp β-action promoter were introduced into channel catfish embryos using CRISPR/Cas9, Tol2 or double electroporation approaches. Three sgRNAs for Elovl2, Δ4 and Δ5/6 donor plasmids were designed to target at chromosome 1, chromosome 2 or chromosome 7 non-coding regions using the CRISPR/Cas9 system. CRISPR/Cas9 and Tol2 approaches were utilized with microinjection and single electroporation delivery methods. Double electroporation delivered donor plasmids to both sperm and fertilized eggs without sgRNA.
A total of 20.8% of microinjected fish using the CRISPR/Cas9 system were positive, double electroporation had a 9.3% integration rate, and there were no positive fish using Tol2 transposon system. Seven fish carrying masu salmon Elovl2 gene and 1 positive fish carrying rabbitfish Δ5/6 gene were identified by PCR and sequencing. CRISPR/Cas9 with microinjection method was more efficient than double electroporation and Tol2. The masu salmon Elovl2 gene was more amenable to gene transfer than the rabbitfish desaturase genes. These fish will play a very important role in building high producing n-3 HUFA channel catfish lines in the future.
Figure 1. PCR analysis of different transgenes integration. A: Masu salmon Elovl2 gene. B: Rabbitfish Δ5/6 gene. M: marker; WT: wild type; C: positive control; H2O: negative control
