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

SIMPLE SUMMARY: Sturgeons, also called archaic giants, are critically endangered fish species due to overfishing for caviar and interference in their natural habitats. Some sturgeon species have life spans of over 100 years and sexual maturity is attained between 20 to 25 years. Sterlet (Acipenser ruthenus) has fastest reproductive cycle; thus, this species can be used for surrogate production in sturgeons. Primordial germ cells are the origin of all germ cells in developing embryos. Dnd1 is essential for formation and migration of primordial germ cells and its inactivation results in sterility in fish. In our study, we have used a cutting-edge genome editing technology known as CRISPR/Cas9 to knockout dnd1 and to prepare a sterile sterlet host. CRISPR/Cas9 knocked-out embryos lacked primordial germ cells and can be used as a sterile host for surrogate production in sturgeons. 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.