Method for producing an animal comprising a germline genetic modification

Abstract

The present invention relates to methods for producing a non-human animal, such as an avian, comprising a targeted germline genetic modification, the method comprising: (i) delivering a programmable nuclease to sperm; (ii) fertilizing an ovum with the sperm, and (iii) generating the animal from the ovum, wherein the nuvlease introduces the genetic modification into DNA of the prem and/or the ovum.

1 . A method for producing a non-human animal comprising a targeted germline genetic modification, the method comprising:
 (i) delivering a programmable nuclease to sperm;   (ii) fertilizing an ovum with the sperm, and   (iii) generating the animal from the ovum,   
       wherein the nuclease introduces the genetic modification into DNA of the sperm and/or the ovum. 
     
     
         2 . The method of  claim 1  which further comprises screening the animal obtained from step (iii) for the germline genetic modification. 
     
     
         3 . The method of  claim 1  or  claim 2 , wherein the nuclease is delivered to the sperm in a composition comprising a transfection agent. 
     
     
         4 . The method according to any one of  claims 1  to  3 , wherein the nuclease is selected from a: RNA-guided engineered nuclease (RGEN), transcription activator-like nuclease (TALEN) and zinc-finger nuclease (ZFN). 
     
     
         5 . The method of  claim 4 , wherein the nuclease is a RNA-guided engineered nuclease (RGEN). 
     
     
         6 . The method according to any one of  claims 2  to  5 , wherein the transfection agent comprises a cationic lipid. 
     
     
         7 . The method of  claim 6 , wherein the transfection agent comprises a monovalent cationic lipid selected from one or more of DOTMA (N-[1-(2.3-dioleoyloxy)-propyl]-N,N,N-trimethyl ammonium chloride), DOTAP (1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane), DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) and DDAB (dimethyl dioctadecyl ammonium bromide). 
     
     
         8 . The method of  claim 6  or  claim 7 , wherein the transfection agent comprises a polyvalent cationic lipid selected from one or more of DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate) and DOSPER (1,3-dioleoyloxy-2-(6carboxy spermyl)-propyl-amid, TMTPS (tetramethyltetrapalmitoylspermine), TMTOS (tetramethyltetraoleylspermine), TMTLS (tetramethlytetralaurylspermine), TMTMS (tetramethyltetramyristylspermine) and TMDOS (tetramethyldioleylspermine). 
     
     
         9 . The method according to any one of  claims 1  to  8 , wherein the genetic modification results in reduced expression of one or more genes and/or proteins in the animal and/or progeny thereof. 
     
     
         10 . The method according to any one of  claims 1  to  9 , wherein the genetic modification is a deletion, substitution or an insertion. 
     
     
         11 . The method of  claim 10 , wherein the insertion is a transgene. 
     
     
         12 . The method according to any one of  claims 1  to  11 , wherein step (ii) comprises artificially inseminating the sperm into a female animal. 
     
     
         13 . The method according to any one of  claims 1  to  12 , wherein at least 10% of the animals produced using the method comprise the targeted germline genetic modification. 
     
     
         14 . The method according to any one of  claims 1  to  13 , wherein all cells of the animal comprise the targeted germline genetic modification. 
     
     
         15 . The method according to any one of  claims 1  to  14 , wherein the animal is heterozygous for the targeted germline genetic modification. 
     
     
         16 . The method according to any one of  claims 1  to  15 , wherein the targeted germline genetic modification modifies a trait of the animal. 
     
     
         17 . The method according to any one  claims 1  to  16 , wherein the animal is an avian. 
     
     
         18 . The method of  claim 17 , wherein the avian is selected from a chicken, duck, turkey, goose, bantam or quail. 
     
     
         19 . An animal produced by the method of any one of  claims 1  to  18 , or progeny thereof. 
     
     
         20 . Sperm or an ovum produced by the animal of  claim 19 . 
     
     
         21 . A method for producing a genetically modified animal, the method comprising:
 (i) crossing a first animal of  claim 19  with a second animal of the same species, and   (ii) selecting progeny comprising the targeted germline genetic modification.   
     
     
         22 . The method of  claim 21  which comprises selecting progeny which are homozygous for the targeted germline genetic modification. 
     
     
         23 . The method of  claim 22 , wherein the first and second animal are heterozygous for the targeted germline genetic modification. 
     
     
         24 . An animal produced by the method according to any one of  claims 21  to  23 . 
     
     
         25 . A method of producing food, the method comprising:
 (i) obtaining the animal of  claim 19  or  claim 24 , and   (ii) producing food from the animal.   
     
     
         26 . The method of  claim 25 , wherein the method comprises harvesting meat and/or eggs from the animal.