Fig. 1

Ribonucleoprotein (RNP) complex transfection in RTgutGC cells. a Overview of the ribonucleoprotein (RNP) complex preparation. Alt-R®crRNA and Alt-R®tracrRNA-ATTO™ 550 are combined at equimolar concentration. Once the duplex crRNA and tracrRNA is formed, Streptococcus pyogenes Cas9 protein is added in order to form the RNP complex. b Overview of the CRISPR/Cas9 gene editing strategy workflow. Briefly, RNP complexes targeting rainbow trout cyp1a1 were transfected in RTgutGC cells via electroporation using the NEPA21 electroporator. Following 48 h of recovery, transfected RTgutGC cells were sorted via FACS using the ATTO™ 550 signal and incubated in a 96 well plate. T7 endonuclease assay and ICE analysis were performed in order to evaluate the overall gene editing efficiency and the nature of the CRISPR/Cas9-induced mutations, respectively. Finally, clonal cell isolation was obtained through low cell density seeding and colony isolation using cloning cylinders. Created with https://biorender.com