Effects of CRISPR/Cas9 dosage on TICAM1 and RBL gene mutation rate, embryonic development, hatchability and fry survival in channel catfish

Ahmed Elaswad, Karim Khalil, Zhi Ye, Zhanjian "John" Liu, Shikai Liu, Eric Peatman, Ramjie Odin, Khoi Vo, David Drescher, Kamal Gosh, Guyu Qin, William Bugg, Nathan Backenstose, Rex Dunham

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Abstract

The current study was conducted to assess the effects of microinjection of different dosages of guide RNA (gRNA)/Cas9 protein on the mutation rate, embryo survival, embryonic development, hatchability and early fry survival in channel catfish, Ictalurus punctatus. Guide RNAs targeting two of the channel catfish immune-related genes, toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) and rhamnose binding lectin (RBL) genes, were designed and prepared. Three dosages of gRNA/Cas9 protein (low, 2.5 ng gRNA/7.5 ng Cas9, medium, 5 ng gRNA/15 ng Cas9 and high, 7.5 ng gRNA/22.5 ng Cas9) were microinjected into the yolk of one-cell embryos. Mutation rate increased with higher dosages (p < 0.05). Higher dosages increased the mutation frequency in individual embryos where biallelic mutations were detected. For both genes, microinjection procedures increased the embryo mortality (p < 0.05). Increasing the dosage of gRNA/Cas9 protein increased the embryo mortality and reduced the hatching percent (p < 0.05). Embryonic development was delayed when gRNAs targeting RBL gene were injected. Means of fry survival time were similar for different dosages (p > 0.05). The current results lay the foundations for designing gene editing experiments in channel catfish and can be used as a guide for other fish species.

Original languageEnglish (US)
Article number16499
JournalScientific Reports
Volume8
Issue number1
DOIs
StatePublished - Dec 1 2018

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Elaswad, A., Khalil, K., Ye, Z., Liu, Z. J., Liu, S., Peatman, E., Odin, R., Vo, K., Drescher, D., Gosh, K., Qin, G., Bugg, W., Backenstose, N., & Dunham, R. (2018). Effects of CRISPR/Cas9 dosage on TICAM1 and RBL gene mutation rate, embryonic development, hatchability and fry survival in channel catfish. Scientific Reports, 8(1), [16499]. https://doi.org/10.1038/s41598-018-34738-4