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

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.

CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome

“…Thus, tissue patterns of expression may or may not be the same when the F 1 generation is produced. However, when our laboratory used CRISPR/Cas9 to produce knockouts without accompanying gene insertion, mosaicism was low and similar among all tissues evaluated 9 .…”

“…Some studies have successfully obtained large genomic deletion using dual guide RNAs (gRNAs) in mammalian cells and animal models such as mouse and zebrafish 17,18 . Deletion mutations of up to 900 bp were recently achieved in channel catfish targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) using a single guide RNA 9 . Moreover, Li et al 8 reported that the CRISPR/Cas9 system could effectively generate desirable non-coding sequence mutants in Nile tilapia (Oreochromis niloticus).…”

The Impact of CRISPR-Cas9 on Age-related Disorders: From Pathology to Therapy

“…The mutation frequency of the embryos increased with greater dosages of gRNA/Cas9 [194]. Higher dosages of gRNA/Cas9 also resulted in decreased hatching percentage and increased mortality [194]. More research is needed to elucidate the relationship between CRISPR-Cas9 dosage and off-target effects.…”

“…The dosage of Cas9 has been demonstrated to alter both its specificity and kinetics, with a 2-fold drop in on-target efficiency alongside a 7-fold increase in the specificity ratio when the Cas9 dosage is decreased by 5-fold during plasmid transfection [ 193 ]. Additional study investigated the possibility of a dose-dependent effect of the gRNA with the Cas9 protein on channel catfish ( Ictalurus punctatus) mutation rate and embryonic survival [ 194 ]. The mutation frequency of the embryos increased with greater dosages of gRNA/Cas9 [ 194 ].…”

“…Additional study investigated the possibility of a dose-dependent effect of the gRNA with the Cas9 protein on channel catfish ( Ictalurus punctatus) mutation rate and embryonic survival [ 194 ]. The mutation frequency of the embryos increased with greater dosages of gRNA/Cas9 [ 194 ]. Higher dosages of gRNA/Cas9 also resulted in decreased hatching percentage and increased mortality [ 194 ].…”