TRANSGENE DELIVERY STRATEGIES TO INTEGRATE CATHELICIDIN GENE IN THE CHANNEL CATFISH GENOME BY CRISPR/CAS9 KNOCK-IN SYSTEM

CRISPR/Cas9-based gene knock out in mammalian cells, particularly in teleosts  has proven to be very efficient in regards to mutation rates , but precise insertion of exogenous DNA  or gene knock-in  via the homology directed repair pathway remains elusive.   While microinjection is  more routinely used to introduce CRISPR components into fish embryos, it is labor-intensive and time-consuming, as embryos need to be injected individually. An alternative to microinjection is electroporation, which uses a rapid and high-voltage electric pulse to deliver  DNA into embryos.  Here, we utilized single electroporation,  where  CRISPR components were co-delivered with the donor DNA into fertilized eggs, and double electroporation,  where  both sperm and fertilized eggs received the  CRISPR components and donor DNA .   In this study, we  designed double-stranded DNA constructs driven by zebrafish ubiquitin (dsDNA Ubi 40ng/µl) and carp β -actin (dsDNA β-actin 40ng/µl) promoters,  as well as plasmid DNA construct driven by  zebrafish ubiquitin promoter  (plasmid Ubi 50ng/µl ) carrying cathel icidin gene, a disease-resistance gene derived from  the American alligator .  

We succeeded in integrating with high efficiency an exogenous cathelicidin gene into chromosome 1 of channel catfish genome.  As shown in Table 1 and Figure 1 , highest integration rates were found using microinjection, both in  dead fry with plasmid

Ubi as donor DNA (61.5%) as well as in alive fingerlings with sdDNA Ubi donor (31.8 %).  Additionally ,  electroporation also proved to be an efficient method  to deliver transgene as indicated  by 44.4 % integration rate in dead fry and 12.5 % in alive fish using single electroporation technique.

Overall,  we have successfully generated a CRISPR knock-in transgenic channel catfish carrying cathelicidin gene using microinjection and electroporation strategies.  In general, integration rates were higher in dead fry, indicating either off-target effects or pleiotropic effects. Additionally, we may be targeting a sensitive area of the genome.