Nanos knock-out that ablates germline cells

Abstract

The present invention provides livestock animals and methods to create recipient animals for spermatogonial stem cell transplantation through modulation of the NANOS gene. In one embodiment genome editing issued to create animals with insertions or deletions (indels) that inactivate or otherwise modulate NANOS gene activity so that resulting males lack functional germ cells yet retain functional somatic cells, and females are fertile. These males can then be transplanted with donor spermatogonial stem cells and used for breeding.

What is claimed is: 
     
         1 . A genetically edited livestock animal comprising at least one edited chromosomal sequence encoding a NANOS protein. 
     
     
         2 . The genetically edited livestock animal of  claim 1 , wherein the edited chromosomal sequence is inactivated, modified, and/or comprises an integrated or deleted sequence. 
     
     
         3 . The genetically edited livestock animal of  claim 1 , wherein the edited chromosomal sequence is inactivated such that little or no functional NANOS protein activity is produced and said animal produces reduced or no germline cells but retains somatic cell function. 
     
     
         4 . The genetically edited livestock animal of  claim 1 , wherein the edited chromosomal sequence comprises no exogenously introduced sequence. 
     
     
         5 . The genetically edited livestock animal of  claim 1  wherein said NANOS protein is selected from the group consisting of NANOS1, NANOS2, or NANOS3. 
     
     
         6 . The genetically edited livestock animal of  claim 1 , wherein the NANOS protein is NANOS2. 
     
     
         7 . The genetically edited livestock animal of  claim 1 , further comprising a conditional knock-out system for conditional expression of the NANOS protein. 
     
     
         8 . The genetically edited livestock animal of  claim 1 , wherein the edited chromosomal sequence comprises an integrated reporter sequence. 
     
     
         9 . The genetically edited livestock animal of  claim 1 , wherein the animal is heterozygous or homozygous for the at least one edited chromosomal sequence. 
     
     
         10 . The genetically edited animal of  claim 1 , wherein the animal is chosen from beef cattle, dairy cattle, pigs, sheep, goats, horses, mules, asses, buffalo, camels, chickens, turkeys, ducks, geese, guinea fowl, and squabs. 
     
     
         11 . The genetically edited animal of  claim 1 , wherein the animal is pig or bovine. 
     
     
         12 . The genetically edited animal of  claim 11  wherein said animal is a pig. 
     
     
         13 . The genetically edited animal of  claim 12  wherein said animal includes an edited NANOS2 gene including SEQ ID NOS: 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 45, 46, 47, 48, 49, 50, 51, 52, 56, 57, 58, 59, 60, 61, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 201, 202, 203, or 204. 
     
     
         14 . The genetically modified animal of  claim 11  wherein said animal is a bovine. 
     
     
         15 . The genetically edited animal of  claim 14  wherein said animal includes an edited NANOS2 gene of SEQ ID NOS: 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 142, 143, 144, 145, 146, 148, 149, 150, 151, or 152. 
     
     
         16 . The genetically edited animal of  claim 1  further comprising transplanted spermatogonial cells to create a recipient the germ line ablated surrogate male. 
     
     
         17 . The recipient germ line ablated surrogate male of  claim 16 . 
     
     
         18 . Sperm from the recipient surrogate male of  claim 17 . 
     
     
         19 . A genetically edited livestock animal cell, the cell comprising at least one edited chromosomal sequence encoding a NANOS protein. 
     
     
         20 . The genetically edited livestock animal cell of  claim 19 , wherein the edited chromosomal sequence is inactivated, modified, or comprises an integrated sequence. 
     
     
         21 . The genetically edited livestock animal cell of  claim 19 , wherein the edited chromosomal sequence is inactivated such that the NANOS protein function is modulated 
     
     
         22 . The genetically edited livestock animal cell of  claim 21 , wherein said NANOS protein is one or more of NANOS1, NANOS2, or NANOS3. 
     
     
         23 . The genetically edited livestock animal cell of  claim 22 , wherein the NANOS protein is NANOS 2. 
     
     
         24 . The genetically edited livestock animal cell of  claim 19 , wherein the cell is heterozygous or homozygous for the at least one edited chromosomal sequence. 
     
     
         25 . The genetically edited livestock animal cell of  claim 19 , wherein the cell is of an animal of one or more of the following: bovine, pig, sheep, goat, horse, mule, ass, buffalo, camel, chicken, turkey, duck, goose, guinea fowl, or squab origin. 
     
     
         26 . The genetically edited livestock animal cell of  claim 25 , wherein the cell is of pig origin. 
     
     
         27 . The genetically edited livestock animal cell of  claim 26  wherein said embryo includes an edited NANOS2 gene of SEQ ID NOS: 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 45, 46, 47, 48, 49, 50, 51, 52, 56, 57, 58, 59, 60, 61, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 201, 202, 203, or 204. 
     
     
         28 . The genetically edited livestock animal cell of  claim 25 , wherein the cell is of bovine origin. 
     
     
         29 . The genetically edited livestock animal of  claim 28  wherein said animal includes an edited NANOS2 gene including SEQ ID NOS 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 142, 143, 144, 145, 146, 148, 149, 150, 151, or 152. 
     
     
         30 . The genetically edited livestock animal cell of  claim 19 , wherein the inactivated chromosomal sequence comprises no exogenously introduced sequence. 
     
     
         31 . The genetically edited livestock animal cell of  claim 19 , further comprising a conditional knock-out system for conditional expression of the NANOS protein. 
     
     
         32 . The genetically edited livestock animal cell of  claim 19 , wherein the edited chromosomal sequence comprises an integrated reporter sequence. 
     
     
         33 . A method of breeding livestock animals comprising:
 collecting sperm from the germ line ablated surrogate male recipient of  claim 17 , and introducing said sperm into a female animal so that said female animal becomes pregnant.   
     
     
         34 . A livestock animal produced by the method of  claim 33 . 
     
     
         35 . The method of  claim 33  wherein said introducing is by artificial insemination. 
     
     
         36 . A method of generating a livestock animal with no functional spermatogonial cells comprising:
 modifying NANOS gene expression so that NANOS protein function is reduced or eliminated.   
     
     
         37 . The method of  claim 36  wherein said animal does not produce a significant amount of spermatogonial cells. 
     
     
         38 . The method of  claim 36  wherein said modifying is by gene editing by use of a TALEN, a zinc finger nuclease, and/or a recombinase fusion protein. 
     
     
         39 . The method of  claim 36  wherein said NANOS gene is edited to include an insertion or deletion which causes inactivation of the gene. 
     
     
         40 . The method of  claim 36  wherein said gene editing modification step includes the use of a NANOS guide RNA, and a polypeptide capable of effecting cleavage or integration of the NANOS target. 
     
     
         41 . The method of  claim 36  wherein said NANOS is NANOS2. 
     
     
         42 . The method of  claim 36  wherein said gene editing modification method is RNA-guided CRISPR/Cas9. 
     
     
         43 . A method of producing a recipient male that serves as a surrogate for donor-derived sperm production for transmission of the donor genotype to offspring via natural or artificial breeding comprising:
 collecting donor SSCs from a desired male donor, and thereafter,   transplanting donor SSCs to a NANOS2−/− recipient male so that spermatogenic colonies are generated.   
     
     
         44 . The method of  claim 43  wherein said NANOS2−/− recipient male includes a gene edited NANOS sequence of SEQ ID NO: 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 45, 46, 47, 48, 49, 50, 51, 52, 56, 57, 58, 59, 60, 61, 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 142, 143, 144, 145, 146, 148, 149, 150, 151, 152, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 201, 202, 203, or 204. 
     
     
         45 . A method of producing livestock animals comprising:
 mating a female livestock animal with sperm from the recipient NANOS2 −/− male of  claim 43 .