Efficient Gene Transfer and Gene Editing in Sterlet (Acipenser ruthenus)

Abstract

The sturgeon (Acipenseriformes) is an important farmed species because of its economical value. However, neither gene transfer nor gene editing techniques have been established in sturgeon for molecular breeding and gene functional study until now. In this study, we accomplished gene transfer and gene editing in sterlet (Acipenser ruthenus), which has the shortest sexual maturation period of sturgeons. The plasmid encoding enhanced green fluorescent protein (EGFP) was transferred into the embryos of sterlet at injection concentration of 100 ng/μL, under which condition high survival rate and gene transfer rate could be achieved. Subsequently, exogenous EGFP was efficiently disrupted by transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease/guide RNA (gRNA), with injection concentrations of 300 ng/μL TALENs, or 100 ng/μL Cas9 nuclease and 30 ng/μL gRNA, respectively, under which condition high survival rate and gene mutation rate could be achieved. Finally, the endogenous gene no tail in sterlet was successfully mutated by Cas9 nuclease/gRNA. We observed the CRISPR-induced no tail mutation, at a high efficiency with the mutant P0 embryos displaying the expected phenotype of bent spine and twisted tail.

Keywords: EGFP; gene editing; gene transfer; no tail; sterlet.

FIGURE 1

(A) The enhanced green fluorescent protein (EGFP) unit in the plasmid pEGFP-C1. (B) Survival rates of EGFP transgenic embryos. A total of 624 embryos were injected. The survival rate was transformed to arcsine square root, then analyzed by ANOVA followed by Student–Newman–Keuls method. Symbols with the same indicate groups that are not significantly different. (C) Detection of EGFP transgene by PCR. M represented marker, lane 1–10 represented 10 individuals, and N represented negative control.

FIGURE 2

(A) Detection of EGFP by fluorescence in embryos at 2 dpf. (B) Detection of EGFP by fluorescence in embryos at 8 dpf.

FIGURE 3

Disruption of EGFP by transcription activator-like effector nucleases (TALENs) in sterlet. A total of 532 embryos were injected. (A) Detection of mutation by PAGE. (B) Survival rate of injected embryos. (C) Mutation rate at target site. The survival rate and mutation rate were transformed to arcsine square root, then analyzed by ANOVA followed by Student–Newman–Keuls method. Symbols with the same indicate groups that are not significantly different.

FIGURE 4

Disruption of EGFP by CRISPR/Cas9 in sterlet. A total of 682 embryos were injected with Cas9 mRNA, and 625 were injected with Cas9 nuclease. (A) Induced by Cas9 mRNA. (B) Induced by Cas9 nuclease. (C) Survival rate of injected embryos. (D) Mutation rate at target site. The survival rate and mutaion rate were transformed to arcsine square root, then analyzed by ANOVA followed by Student–Newman–Keuls method. Symbols with the same indicate groups that are not significantly different.

FIGURE 5

Detection of EGFP disruption by fluorescence.

FIGURE 6

(A) Detection of ntl gene mutation by PAGE. (B) Mutation rate (Ht%) of individuals. (C) Phenotype of ntl disruption.