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.

Dnd1 Knockout in Sturgeons By CRISPR/Cas9 Generates Germ Cell Free Host for Surrogate Production

“…Our present study was aimed to disrupt the Ardnd1 to achieve sterility in sterlet ( Acipenser ruthenus ) to prepare a host for surrogate production in critically endangered sturgeon species. This study is consistent with Chen et al, [53], suggesting that Cas9 endonuclease can be efficiently applied in sturgeon having a higher ploidy level such as hexaploid or octoploid genomes.…”

“…In fish model species, zebrafish, the CRISPR/Cas9 research with codon optimization of Cas9 mRNA showed induction of mutation with 75 to 99% rates in endogenous genes [52]. However, same method does not induce more than 55% mutation in sterlet, which might be due to different characteristics of embryos such as size, incubation temperature, cleavage pattern and ploidy level [53]. Interestingly, in sterlet when Cas9 protein instead of Cas9 mRNA was injected, mutation and survival rate of embryos increased by more than 90% [53].…”

“…However, same method does not induce more than 55% mutation in sterlet, which might be due to different characteristics of embryos such as size, incubation temperature, cleavage pattern and ploidy level [53]. Interestingly, in sterlet when Cas9 protein instead of Cas9 mRNA was injected, mutation and survival rate of embryos increased by more than 90% [53]. Our present study was aimed to disrupt the Ardnd1 to achieve sterility in sterlet ( Acipenser ruthenus ) to prepare a host for surrogate production in critically endangered sturgeon species.…”

Applied Molecular Cloning: Present and Future for Aquaculture

“…Atlantic salmon dnd, tyr, slc24a5 (C) [60,61] Sturgeon dnd (C) [62] Atlantic killifish ahr2 (C) [63] Tilapia fox12a, c yp19a1 a, dmrt1, nanos, gsdf, sf-1/nr5a, mstn (C); rspo1, fox12a, c yp19a1 a (T) [64][65][66][67][68][69] Cavefish oca2 (T) [70] Channel catfish lh (Z) [71] Chinese lamprey slc24a5 (C) [ Medaka dnd (M); fox13, dmy, dmc1, fshb, gnrh1 (T); gsdf (Z) [110][111][112][113][114] Red sea bream mstn (C) [115] Rice field eel dmrt1, foxl2, cyp19ala (T) [116] Rohu tlr22 (C) [117] Starlet and sturgeon Ntl, dnd (T & C) [118,119] M, morpholino; Z, zinc finger nuclease (ZFN); T, transcription activator-like effector nucleases (TALEN), C, clustered regularly interspaced short palindromic repeats (CRISPR).…”

Biotechnological innovation in genetic breeding and sustainable green development in Chinese aquaculture

“…自此, 基因组编辑在多种养殖鱼类中得到了应用, 如虹 鳟 [151] 、罗非鱼 [152] 、鲤 [153] 、大西洋鲑 [154] 、斑点叉尾 鮰 [155] 、黄鳝 [156] 、半滑舌鳎 [157] 和小体鲟 [158] 等. 基因 组编辑技术将在水产养殖动物基因功能解析和经济性 状精准设计育种中发挥重要作用 [102,145,149] .…”