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

Abstract

Sturgeons also known as living fossils are facing threats to their survival due to overfishing and interference in natural habitats. Sterlet (Acipenser ruthenus) due to its rapid reproductive cycle and small body size can be used as a sterile host for surrogate production for late maturing and large sturgeon species. Dead end protein (dnd1) is essential for migration of Primordial Germ Cells (PGCs), the origin of all germ cells in developing embryos. Knockout or knockdown of dnd1 can be done in order to mismigrate PGCs. Previously we have used MO and UV for the aforementioned purpose, and in our present study we have used CRISPR/Cas9 technology to knockout dnd1. No or a smaller number of PGCs were detected in crispants, and we also observed malformations in some CRISPR/Cas9 injected embryos. Furthermore, we compared three established methods to achieve sterility in sterlet, and we found higher embryo survival and hatching rates in CRISPR/Cas9, UV and MO, respectively.

Delivery of Iron Oxide Nanoparticles into Primordial Germ Cells in Sturgeon

“…Magnetic-activated cell sorting can be applied when IONs are loaded into PGCs and when the fluorescent signal is difficult to trace under tissues. Efforts and results from our research team have been trying to meet the promise that techniques for ‘‘surrogate production’’ in IUCN red-listed sturgeons are becoming more practicable and convenient [35,45,46,47,48,49].…”

Spermatogonial Stem Cells in Fish: Characterization, Isolation, Enrichment, and Recent Advances of In Vitro Culture Systems

“…The decision as to a SSC's self-renewal or differentiation is mediated by cell-cell communication, and in vitro germ cell culture provides a novel platform with which to investigate the regulatory network that determines cell fate. Furthermore, germ cell culture can be combined with gene editing techniques such as clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated(Cas) for germ-line transmission, cell transplantation, nuclear transfer, and in vitro spermatogenesis [6][7][8][9]. In the following section, we will review fish spermatogonial cell morphology, distribution, identification, and niche, and the endocrine and paracrine regulation of spermatogenesis.…”

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).…”