Gene editing of reproductive hormones to reduce fertility in ictalurus punctatus

Abstract

Disclosed are fish with impaired reproductive capacity and methods and compositions for producing the same. The reproductively impaired fish may include catfish, such as Ictalurus punctatus . The disclosed methods may be generally useful for mitigating environmental impact of escaped genetically engineered fish.

1 . A method for reducing fertility in a fish comprising modifying the DNA sequence of said fish's luteinizing hormone, gonadotropin-releasing hormone, or follicle-stimulating hormone gene. 
     
     
         2 . The method of  claim 1  wherein the DNA sequence is modified using at least one DNA nuclease. 
     
     
         3 . The method of  claim 2  wherein the DNA nucleases are a zinc finger nuclease, a TALEN nuclease, a CRISPR nuclease, or a combination of zinc finger nucleases, TALEN nucleases, or CRISPR nucleases. 
     
     
         4 . The method of  claim 1  wherein the modification to said fish's DNA¬¬ comprises deletions, insertions, base changes, or combinations of any one of these in the DNA sequence. 
     
     
         5 . A method for inducing modifications to a nucleic acid sequence in a living cell comprising electroporating at least two times to deliver at least one plasmid encoding at least one DNA nuclease. 
     
     
         6 . The method of  claim 5  wherein at least one DNA nuclease is a zinc finger nuclease, a TALEN nuclease, a CRISPR nuclease, or a combination of zinc finger nucleases, TALEN nucleases, or CRISPR nucleases. 
     
     
         7 . The method of  claim 6  wherein the DNA nuclease binds to a nucleic acid sequence that is at least 80% similar to SEQ ID NOs:1, 2, 3, 12, 13, 14, 15, or 16. 
     
     
         8 . The method of  claim 5  wherein the modification to the DNA comprises base changes, deletions or insertions in the sequence of said living cell, wherein the DNA comprises the luteinizing hormone, gonadotropin-releasing hormone, or follicle-stimulating hormone gene. 
     
     
         9 . A fish with a modified luteinizing hormone, gonadotropin-releasing hormone, or follicle-stimulating hormone gene such that functional luteinizing hormone, Gonadotropin-releasing hormone, or follicle-stimulating hormone gene product is no longer produced. 
     
     
         10 . The fish of  claim 9  wherein modifications to the fish's luteinizing hormone, gonadotropin-releasing hormone, or follicle-stimulating hormone genes comprise deletions, insertions, base changes, or combinations of any one of these in the native gene sequence. 
     
     
         11 . The fish of  claim 10  wherein the deletions or insertions are in or near nucleic acid sequences with at least 80% similar to SEQ ID NOs:1, 2, 3, 12, 13, 14, 15, or 16. 
     
     
         12 . A DNA vector encoding at least one DNA nuclease that binds to the catfish luteinizing hormone, gonadotropin-releasing hormone, or follicle-stimulating hormone gene sequence. 
     
     
         13 . The vector of  claim 12  wherein the at least one DNA, nuclease is comprised of a zinc finger nuclease, a TALEN nuclease, or a CRISPR nuclease. 
     
     
         14 . The vector of  claim 12  wherein the DNA nuclease binds to a nucleic acid sequence that is at least 80% similar to SEQ ID NOs:1, 2, 3, 12, 13, 14, 15, or 16.