CRISPR/Cas9-mediated precision integration of fat-1 and fat-2 from Caenorhabditis elegans at long repeated sequence in channel catfish (Ictalurus punctatus) and the impact on n-3 fatty acid level

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential for the development and health of human and animals. However, n-3 PUFAs in human diets, aquaculture and livestock feed are mainly from oily marine fishes. To improve n-3 fatty acid levels in channel catfish (Ictalurus punctatus), we generated transgenic channel catfish carrying codon optimized Caenorhabditis elegans fat-1 gene encoding an n-3 fatty acid desaturase and fat-2 gene encoding the delta12 fatty acid desaturase using CRISPR/Cas9 technology. We used a special cocktail design of donor plasmid, utilized mixed HDR, MMEJ and NHEJ repair pathways, and targeted long repeated DNA target sequence (LRS) to improve knock-in (KI) efficiency. Our results showed that two donor DNAs precisely inserted into at least four genomic sites. Detectable on-target KI efficiency of fat-1 was 9.1% (55/605), three times higher than that of fat-2, which might due to the smaller size of the donor fat-1. Sanger sequencing results suggested that HDR was the dominant repair pathway using this method, leading to the highest proportion of on-target KI events. Reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) demonstrated that the fat-1 and fat-2 transgenes had strong expression, and also greatly improved fads6, elovl2 and elovl5 gene expression in liver and muscle tissues of transgenic channel catfish. Fatty acid analysis showed the fat-1-only transgenic channel catfish had the lower levels of 14:0, 16:0, 18:2n-6, total n-6, and the higher levels of DHA, total n-3 and n-3/n-6 ratio than other genotypes including the fat-2-only, co-integration of fat-1 and fat-2, and wild-type control. In particular, DHA, total n-3 and n-3/n-6 ratio were significantly increased by 37%, 19.8% and 25.2%, respectively, in the fat-1-only transgenic fish than that in the wild-type control (P < 0.05). Our fat-1 and fat-2 KI channel catfish hold promise for improving fish nutritional value, reducing fish oil requirement in aquaculture feed, and serving as an alternative source of n-3 PUFAs.