CRISPR/Cas9 - mediated knock-in method can improve the expression and effect of transgene in P1 generation of channel catfish (Ictalurus punctatus)

Abstract

Transgenesis has a wide range of applications in fish breeding and generation of fish models. Previously, it was common to produce transgenic fish by transferring plasmid DNA into early embryos, resulting in random integration, but more precision, targeted integration is possible with CRISPR/Cas9 technology. Channel catfish (Ictalurus punctatus) is an economically important farmed fish in the United States. To make channel catfish an even richer source of nutrients, we produced P1 fish carrying masu salmon (Oncorhynchus masou) elovl2 (OmElovl2) transgene to increase the content of omega-3 (n-3) fatty acids with CRISPR/Cas9-mediated knock-in targeting non-coding region of chromosome 1, and random integration methods. Mosaicism, transgene expression and fatty acids contents were determined. Integration rates of seven-month-old channel catfish generated by CRISPR/Cas9 and random integration methods were 19% and 27.3%, respectively. However, when we tested five tissues including barbel, fin, muscle, liver and kidney of three channel catfish, 13 out of 15 total observations were verified to carry the transgene from three positive P1 fish produced by CRISPR/Cas9 technology. Only five of 15 tissues carrying transgene were detected in three positive P1 fish produced by random integration. Genomic quantitative real-time PCR (qRT-PCR) also suggested that CRISPR/Cas9 transgenic fish had extremely higher average transgene copy numbers than randomly integrated transgenic fish. Additionally, reverse transcription PCR (RT-PCR) and fatty acids analysis revealed that CRISPR/Cas9 P1 fish had strong OmElovl2 transgene expression in most tissues and 20.7% higher DHA than their controls, while randomly integrated P1 fish did not have detectable OmElovl2 expression in any of five tissues detected. There were no significant differences for any fatty acids between transgenic fish produced by random integration and their non-transgenic controls. CRISPR/Cas9 mediated knock-in technology efficiently reduced mosaicism, improved transgene expression and the biological effects of the foreign gene in P1 generation compared to the conventional random integration method. Therefore, transgenesis based on CRISPR/Cas9 technology would shorten breeding programs and improve applications of gene function studies.