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

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

doi:10.1038/s41598-018-34738-4

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.

Highlights

The development of CRISPR/Cas[9] system has made genome editing experiments more efficient, precise, rapid, and economical
The objective of the current study was to assess the effects of microinjection of different dosages of guide RNA (gRNA)/Cas[9] protein on the mutation rate, embryo survival, embryonic development, hatching percent, early fry survival and deformities in channel catfish
We evaluated the effects of microinjection of three dosages of CRISPR/Cas[9] protein targeting the channel catfish TICAM 1 and rhamnose binding lectin (RBL) genes on the mutation rate, embryo mortality, hatching, time to hatch, early fry survival and congenital anomalies

Summary

Introduction

The development of CRISPR/Cas[9] system has made genome editing experiments more efficient, precise, rapid, and economical. Since the CRISPR/Cas[9] technology opened avenues for precise genome editing, it is important to assess the effects of gRNA and Cas[9] on the survival and hatchability of microinjected catfish embryos to allow more effective experimental design for gene editing experiments. Studying such effects would allow accurate estimation of the number of embryos and the amount of gRNA/Cas[9] protein to be injected to generate enough founder fish for functional genomics studies with minimal off-target mutations. Guide RNAs used in this study were designed to target the channel catfish toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene separately

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Results

Conclusion