CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of LT.S. provisional Patent Application No. 62/653,649, filed April 6, 2018, and LT.S. provisional Patent Application No. 62/814,583, filed March 6, 2019, which is hereby incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

[0002] The present application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 5, 2019, is named INX00385WO_SL.txt and is 53,402 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Myostatin (MSTN), is also known as Growth Differentiation Factor-8 (GDF-8) and is part of the Transforming Growth Factor beta (TGFP) Superfamily. Originally described in 1997, (McPherron et al. (1997) Nature 387:83-90) MSTN was shown to negatively regulates muscle mass in mice and mstn knockout mice exhibited excessive growth of skeletal muscle. The so- called“double muscling phenotype” has since been described in many other mammalian species including, cattle, sheep and humans. Myostatin is also expressed in other vertebrates including fish.

[0004] In mammals, myostatin is expressed principally in muscle, but it has been shown to be weakly expressed in adipose tissue, mammary glands, spleen, placenta and heart (Gabillard, et al. (2013) Gen. Comp. Endocrinol. 194:45-54). By contrast, fish myostatin appears to affect a wider range of tissue including brain, red muscle, white muscle, intestine, and gills and may function as a general inhibitor of cell proliferation (Gabillard, J-C et al. (2013) Gen. Comp. Endocrinol. 194:45-54). Growth Differentiation Factor-l l (gdf-11) is a closely related gene to mstn in mammals and is expressed primarily in the brain, and thus it has been hypothesized that gdf-ll and mstn came from the same ancestral gene that was originally expressed in both brain and muscle, and while mammals now have mstn restricted to muscle, fish retain the more ancient form of expression (Gabillard et al). [0005] In fish, mstn genes have three exons of about 300-400 nt separated by two introns (Gabillard et al). Gene duplication over the course of evolution has resulted in two principal orthologs termed mstn-1 and mstn-2 , and in salmonids, a further subdivision of mstn-l and mstn- 2 (termed mstn-la , mstn-lb , mstn-2a and mstn-2b ) (Gabillard et al).

[0006] MSTN has been studied in various fish species including yellow catfish, sea bass, salmon, zebrafish, tilapia, trout, and medaka.

[0007] Chisada et al. made /??.s7//-deficient medaka ( Oryzias latipes) by mutation of mstnC3 l5Y which corresponds to the mutation in Piedmontese cattle that leads to double muscling in cattle (Chisada et al. (2011) Develop. Biol. 359:82-94). The medaka in these studies exhibited muscular hypertrophy and hyperplasia. Medaka, unlike most fish species, has only one myostatin gene. In other studies with species containing multiple myostatin genes, attenuating MSTN expression in fish has had conflicting results. For example, while attenuating MSTN in medaka (by overexpression of a dominant negative version of MSTN) and zebrafish (by inhibiting the activity of the protein) resulted in increased muscle fiber production, there was no effect on muscle weight (Gabillard et al). Studies by Terova et al. ((2012) Mol. Biotechnol. 54(2):673-684) in sea bass failed to show that inhibition of MSTN affected muscular phenotype (Gabillard et al). In other studies relating mstn expression in fasting and refeeding, gave conflicting results such that Gabillard et al. concluded that results obtained from one species of fish cannot easily be extrapolated to other species of fish due to marked divergences of the fish mstn promoters and the experimental conditions and/or physiological stages (Gabillard et al). Further, 350 million years ago, a gene duplication event led to divergence of mstn (yielding what is now termed mstn-l and mstn-2) (Gabillard et al). The expression pattern of mstn-1 and mstn-2 in fish is different with mstn-1 being expressed in brain, muscle and intestine while mstn-2 is apparently restricted to expression in the brain (Gabillard et al). Gabillard et al. hypothesized that mstn may have acquired a muscle-restricted expression in mammals that is not seen in fish. Thus, mstn may not have the same function in fish as it does in mammals. In fact, Gabillard et al. found that there are very few physiological situations in which mstn expression was associated with muscle growth and this strongly suggests that MSTN action in muscle is very limited if it exists (Gabillard et al). [0008] Thus, myostatin’ s role in a given fish species is not predictive of mysotatin influence on muscle in other fish species. Nonetheless, there is a need in the art to increase growth characteristics in food fish.

BRIEF SUMMARY OF THE INVENTION

[0009] The invention provides fish with enhanced growth characteristics. In some embodiments, the fish are farm-raised food fish such as, but not limited to Cichlids, Salmonids, catfish, carp, eels and sardines. In some embodiments, the fish are tilapia, salmon, trout, tuna, eels, catfish, sardines or carp. In some embodiments, the invention provides tilapia with enhanced growth characteristics. In some embodiments the tilapia is a species selected from Oreochromis niloticus (Nile tilapia), Oreochromis aureus (Blue tilapia) and Oreochromis mossambicus (Mozambique tilapia).

[0010] The tilapia of the invention comprises a mutation in the myostatin gene that causes the myostatin gene to be non-functional. The myostatin deficiency may be heterozygous or homozygous. The mutation may be in Exon 1, Exon 2 or Exon 3. In some embodiments, the mutation is selected from the group consisting of the mutations shown in Table 2. In some embodiments of the homozygous mutant tilapia, the mutations are of a mixed form wherein one mutation is selected from a mutation shown in Table 2, and the other allele has a mutation selected from a different mutation shown in Table 2. In some embodiments the genotype of the mixed mutations is one as shown in Table 4.

[0011] In some embodiments, the invention provides a genetically modified tilapia comprising a modification of Exon 1, Exon 2 or Exon 3 of at least one allele of the mstn-1 gene wherein said tilapia produces a reduced amount of myostatin as compared with a wild type tilapia.

[0012] In some embodiments, the modification of Exon 1 comprises a polynucleotide sequence of TGACCA, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

[0013] In some embodiments, the modification of Exon 3 comprises a polynucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:2l, SEQ ID NO:22, or SEQ ID NO:23.

[0014] In some embodiments, the tilapia comprises a modification of Exon 1, Exon 2, or Exon 3 of both alleles of the mstn-1 gene. In some embodiments, the tilapia comprises a modification of Exon 1 of both alleles of the mstn-1 gene. Non-limiting examples include a modification of each allele comprising a polynucleotide sequence independently selected from TGACCA, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0015] In some embodiments, the tilapia comprises a modification of Exon 3 of both alleles of the mstn-1 gene. Non-limiting examples include a modification of each allele comprising a polynucleotide independently sequence selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:2l, SEQ ID NO: 22, and SEQ ID NO:23.

[0016] In some embodiments, the tilapia comprises a modification at Exon 1 and/or Exon 3 of each allele of mstn-1. Non-limiting examples include tilapia with modifications of the mstn-1 alleles independently selected from TGACCA, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: l7, SEQ ID NO:l8, SEQ ID NO: l9, SEQ ID NO:20, SEQ ID NO:2l, SEQ ID NO:22, and SEQ ID NO:23.

[0017] In some embodiments, the tilapia comprise a modification of an mstn-1 allele that results in a modified MSTN-l protein comprising an amino acid sequence of SEQ ID NO: 11, SEQ ID NO:33, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, or SEQ ID NO:40.

[0018] In some embodiments, the tilapia comprise a modification of both alleles of mstn-1 wherein each allele comprises a modification that results in a modified MSTN-l protein produced from the allele comprising an amino acid sequence independently selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:33, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, or SEQ ID NO:40.

[0019] In other embodiments, the mstn-1 gene is knocked out on at least one of the alleles of the tilapia such that at least one copy of the mstn-l gene is absent from the genome of the tilapia. In other embodiments, the tilapia have both alleles knocked out such that both copies of the mstn-l gene is absent from the genome of the tilapia and no myostatin-l is produced.

[0020] The tilapia of the invention exhibit at least one muscle characteristic selected from increased muscle mass, increased muscle weight, increased condition index, increased growth rate, increased fillet weight, increased fillet yield, increased loin weight, increased loin yield decreased feed conversion ratio (FCR). One benefit of the invention is that it enables harvesting of fish at a lower weight but yielding the same fillet size as a larger fish, in order to effectively reduce production turn.

[0021] In some embodiments, the tilapia of the invention has an increase in body weight of at least about 10% to about 100% greater than body weight of wild type tilapia. In some embodiments, the tilapia of the invention has an increase in growth rate that is at least about 10% to about 100% greater than the growth rate of wild type tilapia. In some embodiments, the tilapia of the invention has a condition index of about 3.8 K (g/cm ³ ) to about 6.2 K (g/cm ³ ). In some embodiments, the tilapia of the invention has an increase in fillet weight that is at least about 15% to about 250% greater than fillet weight of wild type tilapia. In some embodiments, the tilapia of the invention has an increase in fillet yield that is at least about 5% to about 150% greater than loin yield of wild type tilapia. In some embodiments, the tilapia of the invention has an increase in fillet yield that is at least about 5% to about 100% greater than loin yield of wild type tilapia. In some embodiments, the tilapia of the invention has an increase in loin yield that is at least about 5% to about 75% greater than fillet yield of wild type tilapia. In some embodiments, the tilapia of the invention has an increase in fillet yield that is at least about 25% to about 75% greater than fillet yield of wild type tilapia.

[0022] The loin weight of the tilapia may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% 45% 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390% or about 400% greater than the loin weight of wild type tilapia. In some embodiments, the tilapia of the invention has an increase in loin weight that is at least about 15% to about 400% greater than loin weight of wild type tilapia. In some embodiments, the tilapia of the invention has an increase in loin weight that is at least about 250% to about 400% greater than loin weight of wild type tilapia. In some embodiments, the tilapia of the invention has an increase in loin weight that is at least about 125% to about 300% greater than loin weight of wild type tilapia. In some embodiments, the tilapia of the invention has an increase in loin weight that is at least about 75% to about 200% greater than loin weight of wild type tilapia. In some embodiments, the tilapia of the invention has an increase in loin weight that is at least about 5% to about 100% greater than loin weight of wild type tilapia. [0023] In further embodiments, the tilapia of the invention may further comprise at least one other genetic modification. In non-limiting examples, the modification is selected from a modification to promote faster growth, temperature tolerance, and salinity tolerance.

[0024] In some embodiments, the modification to promote faster growth is a modification of growth hormone expression to constitutively express growth hormone. The growth hormone may be a tilapia growth hormone or a heterologous growth hormone.

[0025] In some embodiments, the modification to promote temperature tolerance is a modification of fatty acid desaturase expression to increase expression of fatty acid desaturase. The fatty acid desaturase may be a tilapia fatty acid desaturase or a heterologous fatty acid desaturase.

[0026] In some embodiments, the modification to promote salinity tolerance is a modification of polyvalent cation-sensing receptor expression to increase expression of polyvalent cation-sensing receptor. The polyvalent cation-sensing receptor may be a tilapia polyvalent cation-sensing receptor or a heterologous polyvalent cation-sensing receptor.

[0027] The invention also provides methods of producing and breeding tilapia of the invention. In some embodiments, the method produces heterozygous fish with reduced MSTN-l activity or no MSTN-l activity from the modified allele. In some embodiments, the method involves modifying the second allele of mstn-1 to produce a homozygous fish with reduced MSTN-l or no MSTN-l activity. In some embodiments, the reduced MSTN-l activity is 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less of that produced by wild type tilapia.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 shows an alignment of Nile tilapia, Belgian Blue cattle, and Piedmontese cattle MSTN-l/MSTN protein sequences. Natural cattle mutations annotated in black highlighting; Stop codons are noted by and identities among the three species are noted with an asterisks.

[0029] Figure 2: Panel A shows DNA sequence alignment of the mutations in MSTN-l Exon 1 identified in heterozygous Fl mutants; Panel B shows protein sequence alignment (MSTN-l Exon 1 only) based on mutations identified in heterozygous Fl mutants; Panel C shows DNA sequence alignment of the mutations in MSTN-l Exon 3 identified in heterozygous Fl mutants; Panel D shows protein sequence alignment (MSTN-l Exon 3 only) based on mutations identified in heterozygous Fl mutants. Differences between the WT MSTN-l reference sequence and the mutants are shown in bold.

[0030] Figure 3 shows protein sequence alignment (full MSTN-l protein) based on mutations identified in heterozygous Fl mutants. Differences between the WT MSTN-l reference sequence and the mutants are shown in black highlighting with white letters.

[0031] Figure 4 shows tilapia body weight vs age: (A) Average weight; (B) Batch A Genotyped weights; (C) Batch B genotyped weights; (D) Batch C genotyped weights. Horn: homozygous mutant; Het: heterozygous mutant; WT : wildtype.

[0032] Figure 5 shows Batch A data at l82-d.p.f. (A) Individual fish weights; (B) growth rates; (C) Condition Index vs. weight; (D) Condition indices. Horn: homozygous mutant; Het: heterozygous mutant; WT : wildtype.

[0033] Figure 6 shows Batch A fillet results for fillet weight (A) and fillet yield (B).

[0034] Figure 7 shows Batch B data at l48-d.p.f. : (A) Individual fish weights; (B) growth rates; (C) Condition Index vs. weight; (D) Condition indices. Horn: homozygous mutant; Het: heterozygous mutant; WT : wildtype.

[0035] Figure 8 shows Batch C data at 98-d.p.f : (A) Individual fish weights; (B) growth rates; (C) Condition Index vs. weight; (D) Condition indices. Horn: homozygous mutant; Het: heterozygous mutant; WT : wildtype.

[0036] Figure 9 shows Batch A, B and C Growth rates; (A) Batch A; (B) Batch B; (C) Batch C; Horn: homozygous mutant; Het: heterozygous mutant; WT: wildtype.

[0037] Figure 10 shows characteristics of Batches A, B and C: (A) Batch A fillet weight; (B) Batch A fillet yield; (C) Batch C fillet weight; (D) Batch B fillet yield; (E) Batch C fillet weight; (F) Bactch C fillet yield; Horn: homozygous mutant; Het: heterozygous mutant; WT: wildtype.

[0038] Figure 11 shows Batch D characteristics for males and female fish: (A) fillet weight; (B) fillet yield; (C) loin weight; (D) loin yield; Horn: homozygous mutant; Het: heterozygous mutant; WT : wildtype.

[0039] Figure 12 shows photographs of Batch D loins for male fish: Horn: homozygous mutant; Het: heterozygous mutant; WT: wildtype. [0040] Figure 13 shows Batch D characteristics: (A) weekly body weight; (B) study weight gain; (C) study feed conversion ratio (FCR); Horn: homozygous mutant; Het: heterozygous mutant; WT : wildtype.

[0041] Figure 14 shows Batch F characteristics from week 0 to week 6 post-fertilization: (A) weekly body weight; (B) total weight gain; (C) specific growth rate; (D) study feed conversion ratio (FCR); Horn: homozygous mutant; Het: heterozygous mutant; WT: wildtype.

[0042] Figure 15 shows Batch F characteristics from week 0 to week 19: (A) body weight; (B) body length; Horn: homozygous mutant; Het: heterozygous mutant; WT: wildtype.

[0043] Figure 16 shows Batch F characteristics for fish harvested when homozygous fish reached a weight of approx. 850 g: (A) body weight; (B) body length; Horn: homozygous mutant; Het: age-matched, heterozygous mutant; WT: age-matched wildtype.

[0044] Figure 17 shows Batch F characteristics for fillets and loins from fish harvested when homozygous fish reached a weight of about 850 g: (A) fillet weight; (B) fillet yield; (C) loin weight; (D) loin yield; Horn: homozygous mutant; Het: heterozygous mutant; WT: wildtype.

[0045] Figure 18 shows Batch F measurements of fillets and loins from fish harvested when homozygous fish reached a weight of about 850 g: (A) fillet length; (B) fillet width; (C) fillet thickness; (D) loin length; (E) loin width; (F) loin thickness; Horn: homozygous mutant; Het: heterozygous mutant; WT : wildtype.

[0046] Figure 19 shows a photograph of WT fish beside precision-bred fish; Horn: homozygous mutant; WT : wildtype.

DETAILED DESCRIPTION OF THE INVENTION

[0047] This description contains citations to various journal articles, patent applications and patents. These are herein incorporated by reference as if each was set forth herein in its entirety.

[0048] The invention provides genetically engineered fish, such as tilapia, having a disruption of the myostatin gene such that the modified fish exhibit increased muscle mass, increased muscle weight, improved condition index, increased growth rate, increased fillet weight, increased fillet yield and decreased Feed Conversion Ratio (FCR) as compared to wild-type fish.

[0049] As used herein“tilapia” refers to a cichlid fish of the genera Oreochromis, Sarotherodon, and Tilapia. In some embodiments, the tilapia is selected from one or more of Oreochromis niloticus (Nile tilapia), Oreochromis aureus (Blue tilapia) and Oreochromis mossambicus (Mozambique tilapia).

[0050] As used herein, an“ mstn gene” refers to the myostatin genes and includes both the mstn- 1 and mstn-2 genes.

[0051] As used herein, an“MSTN polypeptide” or“MSTN protein” refers to myostatin protein and includes both the MSTN-l and MSTN-2.

[0052] A“nucleic acid” is a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA) (sometimes referred to herein as“polynucleotide,”) both of which may be single-stranded or double-stranded. DNA includes but is not limited to cDNA, genomic DNA, plasmids DNA, synthetic DNA, and semi -synthetic DNA. DNA may be linear, circular, or supercoiled.

[0053] A “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxynbonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester anologs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA- DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double- stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A“recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.

[0054] The term“fragment” of a polynucleotide will be understood to mean a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence identical to the reference nucleic acid. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least 8, 10, 12, 15, 18, 20 to 25, 30, 40, 50, 70, 80, 100, 200, 500, 1000 or 1500 consecutive nucleotides of a nucleic acid according to the invention.

[0055]“Fragment” of a polypeptide according to the invention will be understood to mean a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises, over the entire portion with these reference polypeptides, an identical amino acid sequence. Such fragments may, where appropriate, be included in a larger polypeptide of which they are a part. Such fragments of a polypeptide according to the invention may have a length of 10, 15, 20, 30 to 40, 50, 100, 200 or 300 amino acids.

[0056] A“gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.“Gene” also refers to a nucleic acid fragment that expresses a specific protein or polypeptide, including regulatory sequences preceding (5' noncoding sequences) and following (3' non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its own regulatory sequences.“Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and/or coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A chimeric gene may comprise coding sequences derived from different sources and/or regulatory sequences derived from different sources.“Endogenous gene” refers to a native gene in its natural location in the genome of an organism A “foreign” gene or “heterologous” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A“transgene” is a gene that has been introduced into the genome by a transformation procedure.

[0057]“Heterologous” DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell.

[0058]“Homologous recombination” refers to the insertion of a foreign DNA sequence into another DNA molecule, e.g., insertion of a vector in a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.

[0059] The term“expression,” as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into a protein or polypeptide.

[0060] The terms“modulate” and“modulates” mean to induce, reduce or inhibit nucleic acid or gene expression, resulting in the respective induction, reduction or inhibition of protein or polypeptide production.

[0061] A“protein” is a polypeptide that performs a structural or functional role in a living cell.

[0062] Percentages and other numerical values expressed herein as a series ( e.g ., 10%, 15%, 20%, 25%, 30%, 35% or more) includes any of the ranges among all in the series (e.g., 10% to 15%, 10% to 30%, 15% to 35%, 10% to 35%). The numerical values also include any whole number or tenth of an integer between such values. For example, recited values 1.2, 1.4, 1.6, 1.8 include 1.3, 1.5, and 1.7 and recited values 10%, 20%, 25% includes 11%, 12%, 13%, etc.

[0063] As used herein,“increased muscle mass” means a gain in bulk of muscular tissue. The gain may be due to production of more muscle cells or greater hypertrophy of muscle cells. In the invention, the modified fish exhibit increased muscle mass as compared with wild type fish.

[0064] As used herein,“increased muscle weight” means a gain in weight of lean muscular tissue. In the invention, the modified fish exhibit increased muscle weight as compared with wild type fish.

[0065] As used herein,“improved condition index” means a gain in a measurement of the overall health of a fish by comparing its weight with the typical weight of other fish of the same kind and of the same length.

[0066] As used herein, increased growth rate means a gain in achieving a given size over a period of time. In the invention, the modified fish exhibit increased growth rate as compared with wild type fish. [0067] As used herein, increased fillet weight means a gain in the amount of boneless meat of a fish by weight. In the invention, the modified fish exhibit increased fillet weight as compared with wild type fish prepared in the same way.

[0068] As used herein, increased fillet yield means a gain in the percentage of fillet weight per overall weight of a fish. In the invention, the modified fish exhibit increased fillet yield as compared with wild type fish.

[0069] As used herein, increased loin weight means a gain in the amount of boneless meat above the spine of a fish by weight. In the invention, the modified fish exhibit increased loin weight as compared with wild type fish prepared in the same way.

[0070] As used herein, increased loin yield means a gain in the percentage of loin weight per overall weight of a fish. In the invention, the modified fish exhibit increased loin yield as compared with wild type fish.

[0071] As used herein, MSTN-l refers to the myostatin-l gene product (protein). As used herein, mstn-1 refers to the myostatin gene (nucleic acid encoding myostatin).

[0072] The invention provides for genetically engineered tilapia that have a disruption of the mstn gene or knocked out mstn gene. In some embodiments, the fish may have disruptions of both mstn-1 and mstn-2 genes. The disruptions may be disruptions that result in non-functional MSTN proteins or a total knockout of the mstn gene. The disruptions may be on one or both alleles of mstn-1 and/or mstn-2. As such, the tilapia of the invention may be heterozygous or homozygous for mutations in mstn which effectively reduces or eliminates MSTN in the modified fish. In certain embodiments, the fish are heterozygous for a null mutation of mstn-1. In other embodiments, the fish are homozygous for the same null mutation of mstn-1. In other embodiments, the fish are homozygous for different null mutations of mstn-1. In certain embodiments, the fish are heterozygous for a null mutation of mstn-2. In other embodiments, the fish are homozygous for the same null mutation of mstn-2. In other embodiments, the fish are homozygous for different null mutations of mstn-2. In certain embodiments, the fish are heterozygous for a null mutation of both mstn-1 and mstn-2. In other embodiments, the fish are homozygous for the same null mutation of mstn-1 and for the same null mutation of mstn-2. In other embodiments, the fish are homozygous for different null mutations of mstn-1 and different null mutations of mstn-2. [0073] In still other embodiments, the fish are heterozygous for a mutation of mstn-1 in which the fish produce a reduced amount of MSTN-l, such as 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less of that produced by wild type tilapia. In other embodiments, the fish are homozygous for the same or different mutations in mstn-1 in which the fish produced reduced amount of MSTN-l, such as 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less of that produced by wild type tilapia. In other words, the precision-bred fish produce only 50% of the amount of MSTN-l that wildtype fish produce, 40% of the amount of MSTN-l that wildtype fish produce, 30% of the amount of MSTN-l that wildtype fish produce, etc. In some cases, the fish produce no MSTN-l.

[0074] In still other embodiments, the fish are heterozygous for a mutation of mstn-2 in which the fish produce a reduced amount of MSTN-2, such as 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less of that produced by wild type tilapia. In other embodiments, the fish are homozygous for the same or different mutations in mstn-2 in which the fish produced reduced amount of MSTN-2, such as 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less of that produced by wild type tilapia. In other words, the transgenic fish produce only 50% of the amount of MSTN-2 that wildtype fish produce, 40% of the amount of MSTN-2 that wildtype fish produce, 30% of the amount of MSTN-2 that wildtype fish produce, etc. In some cases, the fish produce no MSTN-2.

[0075] In still other embodiments, the fish are heterozygous for a mutation of mstn-1 and a mutation of mstn-2 in which the fish produce a reduced amount of MSTN-l and MSTN-2, such as 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less of that produced by wild type tilapia. In other embodiments, the fish are homozygous for the same or different mutations in mstn-1 and a mutation of mstn-2 in which the fish produce a reduced amount of MSTN-l, such as 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or less of that produced by wild type tilapia. In other words, the transgenic fish produce only 50% of the amount of MSTN-l and MSTN-2 that wildtype fish produce, 40% of the amount of MSTN-l and MSTN-2 that wildtype fish produce, 30% of the amount of MSTN-l and MSTN-2 that wildtype fish produce, etc. In some cases, the fish produce no MSTN-l or MSTN-2.

[0076] The tilapia of the invention comprise at least one mutation of the mstn-1 gene which may be in Exon 1, Exon 2 or Exon 3 of mstn-1. [0077] In some embodiments, the fish comprise a mutation of the mstn-1 gene in Exon 1. The mutation in Exon 1 is one that truncates or modifies the MSTN-l protein as to be non-functional in the fish. Examples of such mutations are those in mstn-1 with the nucleic acid sequences shown in Table 2 and in Fig. 2A below. The polynucleotide sequence of the mstn-1 mRNA is shown as SEQ ID NO: l and the spliced CDS sequence is shown as SEQ ID NO:32. In some embodiments, the fish comprise a mutation in Exon 1 wherein the polynucleotide sequence contains a mutation shown in as MSTN-l c.70_95del (TGACCA); MSTN-l c.73_93del (SEQ ID NO: 6); MSTN-l c.74_90delinsG (SEQ ID NO: 7); MSTN-l c.75_94delinsTCATCA (SEQ ID NO: 8); or MSTN-l c.75_77delinsACTACTGGCGGAAGGCT (SEQ ID NO: 9) with respect to wild type MSTN-l portion (SEQ ID NO: 5) as shown in FIG. 2A. The resulting amino acid changes with respect to wildtype MSTN-l Exon 1 (SEQ ID NO: 10) are shown in FIG. 2B as MSTN-l c.70_95del (SEQ ID NO: 11); MSTN-l c.73_93del (SEQ ID NO: 12); MSTN-l c.74_90delinsG (SEQ ID NO: 13); MSTN-l c.75_94delinsTCATCA (SEQ ID NO: 14); or MSTN-l c.75_77delinsACTACTGGCGGAAGGCT (SEQ ID NO:l5).

[0078] In some embodiments, the fish comprise a mutation in Exon 3 in which the mutation in Exon 3 is one that truncates or modifies the MSTN-l protein as to be non-functional in the fish. In some embodiments, the fish comprise a mutation in mstn-1 Exon 3 wherein the polynucleotide sequence contains a mutation shown in FIG. 2C with respect to the wildtype portion of the polynucleotide encoding MSTN-l Exon 3 (SEQ ID NO: 16) and are shown as MSTN-l c837_838insAGTTGTCGCTC (SEQ ID NO: 17); MSTN-l c837_84ldel (SEQ ID NO: 18); MSTN-l c837_842del (SEQ ID NO: 19); MSTN-l c837delinsTGAGAACTC (SEQ ID NO:20); MSTN-l c837_839insT (SEQ IDNO:2l); MSTN-l 839_845delCCCGGTG (SEQ ID NO:22); and MSTN-l c[843G>T; 85lG>A] (SEQ ID NO:23). The resulting amino acid changes with respect to wildtype MSTN-l Exon 3 (SEQ ID NO:24) are shown in FIG. 2D as MSTN-l c837_838insAGTTGTCGCTC (SEQ ID NO: 25); MSTN-l c837_84ldel (SEQ ID NO:26); MSTN-l c837_842del (SEQ ID NO:27); MSTN-l c837delinsTGAGAACTC (SEQ ID NO:28); MSTN-l c837_839insT (SEQ IDNO:29); MSTN-l 839_845delCCCGGTG (SEQ ID NO:30); or MSTN-l c[843G>T; 85lG>A] (SEQ ID NO:3 l).

[0079] In some embodiments, as shown in FIG. 3, the fish have an mstn-1 allele resulting from a mutation in Exon 1 and which results in a MSTN-l polypeptide having the amino acid sequence of MSTN-l c.70_95del (SEQ ID NO: l l); MSTN-l c.73_93del (SEQ ID NO:33); MSTN-l c.74_90delinsG (SEQ ID NO: 13); MSTN-l c.75_94delinsTCATCA (SEQ ID NO: 14); or MSTN-l c.75_77delinsACTACTGGCGGAAGGCT (SEQ ID NO: 15). In some embodiments, the fish have an mstn-1 allele resulting from a mutation in Exon 3 and which results in a MSTN- 1 polypeptide having the amino acid sequence of MSTN-l c837_838insAGTTGTCGCTC (SEQ ID NO:34); MSTN-l c837_84ldel (SEQ ID NO:35); MSTN-l c837_842del (SEQ ID NO:36); MSTN-l c837delinsTGAGAACTC (SEQ ID NO:37); MSTN-l c837_839insT (SEQ IDNO:38); MSTN-l 839_845delCCCGGTG (SEQ ID NO:39); or MSTN-l c[843G>T; 85 lG>A] (SEQ ID NO:40) with respect to the wildtype MSTN-l amino acid sequence (SEQ ID NO:2).

[0080] The tilapia of the invention exhibit an increase in at least one muscle characteristic selected from muscle mass, muscle weight, condition index, growth rate, fillet weight, fillet yield, loin weight or loin yield.

[0081] The tilapia of the invention may exhibit an increase in body weight of at least 10% to 100% compared to wild type tilapia. In certain embodiments, the increase in body weight is at least 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to wild type tilapia.

[0082] The tilapia of the invention may exhibit an increase in growth rate of at least 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to wild type tilapia. In some embodiments, the tilapia exhibit growth rate of 40%, 45%, 55% or 60%. The homozygous tilapia may exhibit an average growth rate of about 40% or more.

[0083] The tilapia of the invention has an increased condition index. Heterozygous fish had an average condition index of about 3.8 K (g/cm ³ ) in a range of about 2.9 K (g/cm ³ ) to about 4.9 K (g/cm ³ ). Homozygous fish had an average condition index of about 5.5 K (g/cm ³ ) in a range of about 4.8 K (g/cm ³ ) to about 6.2 K (g/cm ³ ). In some embodiments, the tilapia have a condition index of about 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8 or 7.0 K (g/cm ³ ).

[0084] The tilapia of the invention has an increased fillet weight as compared to wild type tilapia. In some examples, the heterozygous fish had an average fillet weight of 86.13 g which was a 17% increase in fillet weight as compared to wild type tilapia. In some examples, the heterozygous fish had an average fillet weight of 30.73g which was a 16% increase in fillet weight as compared to wild type tilapia. In some embodiments, the upper range in increase in fillet weight in heterozygotes was about 53 g or about twice the fillet weight of wild type, age- matched tilapia. In some embodiments, the upper range in increase in fillet weight in heterozygotes was about 140 g or about twice the fillet weight of wild type, age-matched tilapia taken at a heavier weight. In some embodiments, the homozygous tilapia of the invention had an average fillet weight of 40.29 g which was a 54% increase as compared to wild type, age- matched tilapia. In some embodiments, the homozygous tilapia of the invention had an average fillet weight of 204.4 g which was a 177% increase as compared to wild type, age-matched tilapia taken at a heavier weight. In some embodiments, the upper range of increase in fillet weight in homozygotes was about 58 g which is about 2.2 times the fillet weight of wild type, age-matched tilapia. In some embodiments, the upper range of increase in fillet weight in homozygotes was about 342 g which is about 4.6 times the fillet weight of wild type, age- matched tilapia taken at a heavier weight. Thus in some embodiments, the increase in fillet weight of the tilapia of the invention is 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 245%, 250%, or more.

[0085] The fillet yield of the tilapia of the invention was increased as compared to wild type tilapia. In some embodiments, the average fillet yield in homozygous fish was 52.12% which was an increase of 27% over wild type, age-matched tilapia. In some embodiments, the average fillet yield in homozygous fish was 53.33% which was an increase of 63% over wild type, age- matched tilapia taken at a heavier weight. In some embodiments, the upper range of the fillet yield in homozygous fish was about 55% (about a 35% increase over wild type, age-matched tilapia). In some embodiments, the upper range of the fillet yield in homozygous fish was about 68% (109% increase over wild type, age-matched tilapia taken at a heavier weight). In the early growth studies, the upper range of fillet yield in heterozygotes was about 53% while the average fillet weight did not differ from wild type, age matched tilapia. However, in later stages of growth, the upper range of fillet yield in heterozygotes was about 43% while the average fillet weight was 37% (about a 14% increase over wild type, age-matched tilapia). Thus, in some embodiments, the increase in fillet yield of the tilapia of the invention over wild type is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or greater.

[0086] The tilapia of the invention has an increased loin weight as compared to wild type tilapia. In some example, the homozygous tilapia of the invention had an average loin weight of 99 g which was a 302% increase as compared to wild type tilapia. The upper range of increase in loin weight in homozygotes was about 133 g which is about 5.4 times the loin weight of wild type tilapia. Thus in some embodiments, the increase in loin weight of the tilapia of the invention is 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 210%, 215%, 220%, 225%, 230%, 235%,

240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 295%, 300%,

305%, 310%, 315%, 320%, 325%, 330%, 335%, 340%, 345%, 350%, 355%, 360%, 365%,

370%, 375%, 380%, 385%, 390%, 395%, 400% or more.

[0087] The loin yield of the tilapia of the invention was increased as compared to wild type tilapia. The average loin yield in homozygous fish was 21.48% which was an increase of 120% over wild type tilapia. The upper range of the loin yield in homozygous fish was about 25% (156% increase over wild type. Thus, in some embodiments, the increase in fillet yield of the tilapia of the invention over wild type is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150% or greater.

Additional Traits

[0088] The invention also provides fish with myostatin gene knockout with one or more additional engineered traits. Such traits may include at least one, but not limited to the following: increased growth rate, saline tolerance and high or low temperature tolerance.

[0089] Genetic modification for increased growth rate has been shown in salmon and described in U.S. Patent No. 6,698,844 to Hew (incorporated herein by reference). One may use a tilapia growth hormone under the control of a promoter to express the growth hormone in tilapia, or a growth hormone from another species to engineer into a myostatin knockout tilapia to increase the tilapia’ s growth and obtain the benefit of the myostatin knockout traits. [0090] It has also been shown that certain receptors in salmon allow fish to sense and adapt to changing ionic concentrations in surrounding water. Receptors, such as the polyvalent cation sensing receptor (PVCR), and methods of introducing nucleic acids for expressing PVCR in fish, including tilapia, are described in WO0231149 (incorporated herein by reference). One may use a tilapia PVCR or a PVCR from another species to engineer into a myostatin knockout tilapia to increase the tilapia ability to tolerate and adapt to changing salinities in the environment.

[0091] It has also been shown that the fatty acid desaturase gene may be expressed in fish to increase fatty acid content to render the fish more tolerant of low temperatures. U.S. patent Application No. US2008/0320610 (incorporated herein by reference) discloses methods of expressing the fatty acid desaturase gene in fish including tilapia. One may use a fatty acid desaturase from tilapia or from another species to engineer into a myostatin knockout tilapia to increase the tilapia’ s tolerance of colder temperatures.

Methods of making MSTN-1 KO fish

[0092] The tilapia mutations may be introduced by any means known in the art. Tilapia become sexually mature in five months, and female tilapia are ready to spawn every two weeks.

Tol2 Mutagenesis

[0093] Transgenic and knockout tilapia with altered or no expression of mstn-1 and/or mstn-2 may be generated using the Tol2 system (Fujimua, K. and T.D. Kocher (2011) Aquaculture 3 l9(3-4):342-346). Briefly, a plasmid may be constructed to contain minimal elements for Tol2 transposition as well as cassette for integration into the tilapia genome (such as, for example, by incorporating a modified mstn-1 or a reporter gene (e.g., green fluorescent protein (GFP)) flanked by polynucleotides that are complementary to regions that flank the natural mstn-1 gene. The plasmid may be injected into tilapia microinjecting fertilized eggs at the one-cell stage along with tol2 transposase mRNA and raising the embryos to adult fish. This technique may be used for modifying or knocking out mstn-1 and/or mstn-2. Mutations or knockout of the mstn-1 or mstn-2 may be confirmed by sequencing.

CRE/Lox and FLP/FLT

[0094] Transgenic and knockout tilapia may also be generated using homologous recombination using the CRE/Lox system such as that used for zebrafish ( Danio rerio ) (Dong J. and G.W. Stuart (2004) Methods Cell Biol 77:363-379; Pan X. et al. (2005) Trans. Res. 14:217-223). Additional tools for making transgenic fish include the use of the FLP/FRT site-specific recombination system (Wong, A.C. et al. (2011) Transgenic Res. 20:409-415).

Knock out by insertional mutagenesis

[0095] In one example of producing knockout tilapia, a modified gene construct is produced to replace the existing allele or fragment of the allele of wild type tilapia. A complete knockout of the mstn-1 gene can also be made by techniques known in the art. A polynucleotide, such as a selectable marker, flanked by tilapia polynucleotide sequences that are homologous to the region that flank the endogenous mstn-1 gene can be made, for example to replace and knockout the endogenous mstn-1 gene on an allele. Cross breeding the Fl generation can lead to a homozygous knockout of the gene on both alleles.

Gene Editing

[0096] Gene editing strategies may also be used to introduce targeted mutations in Tilapia genes to create fish with altered expression of genes or knockouts using various editing strategies such as Transcription Activator-Like Effector Nucleases (TALENs) (Li, M.H. et al. (2013) Endocrinol. 154: 4814-4825); Zinc Finger Nucleases (ZFN) (Doyon Y. et al. (2008) Nat. Biotechnol. 26:702-708); and recently, using CRISPR/Cas9 (Li, M. et al. (2014) Genetics 197:591-599).

Homology-Directed Repair

[0097] Embryos of fish may be injected with polynucleotide constructs designed to introduce a selectable marker (such as, for example but not by way of limitation, a neomycin resistance gene (NEO)) into an exon of a gene of interest in the fish genome by homologous recombination. That is, the selectable marker sequence is flanked by nucleic acid sequence that is complementary to the region of DNA to be targeted. Here, sequences of mstn-l may flank the NEO gene such that the NEO gene would be inserted into the locus for mstn-l by homologous recombination and thereby disrupt the mstn-l gene and function. In this way, the mstn-l gene could be disrupted or even knocked out entirely by using flanking sequences that are complementary to the flanking sequence of the mstn-l gene in the genome.

[0098] In some embodiments, homology directed repair may be used in which a first targeted endonuclease is introduced into a fish cell or embryo at a desired site in which two homology- directed repair templates are designed to flank a target region on a chromosome for disruption. Thus, a first homology directed repair nuclease is introduced along with a first homology directed repair template homologous to a first target site sequence on the chromosomal DNA. A second targeted endonuclease directed to a second target chromosomal DNA site is also introduced along with the second homology directed repair template (homologous to the sequence of the second target site on the chromosome). In this case, the first homology directed repair sequence replaces the native sequence on the chromosome and the second homology directed repair template sequence replacing the native sequence on the chromosome and introduces the designed change of sequence on the chromosome of the targeted area.

[0099] For example, one may employ meganucleases to effectuate double strand breaks and meganuclease or an enzyme derived from such a meganuclease, such as a synthetic meganuclease. Non-limiting examples of such meganucleases, include I-Ceul, I-Crel, I-Chul, I- Csml, I-Dmol, I-Panl, I-Scel, I-Scell, I-SceIII, I-SceIV, F-Scel, F-Scell, PI-Aael, PI-Apel, PI- CeuI, PI-Cirl, PI-Ctrl, PI-Dral, PI-MavI, PI-Mfll, PI-Mgol, PI-Mjal, PI-Mkal, PI-Mlel, PI-MtuI, PI-MtuHI, PI-PabIII, Pl-Pful, PI-PhoI, Pl-Pkol, PI-PspI, PI-Rmal, Pl-Scel, PI-SspI, PI-Tful, PI- Tlil, PI-TliII, PI-TspI, PI-TspII, PI-BspI, PI-Mchl, PI-Mfal, PI-Mgal, PI-MgaII, PI-MinI, PI- Mmal, PI-MshI, PI-MsmII, PI-MthI, PI-TagI, RI-ThyII, I-Ncrl, I-NcrII, I-PanII, I-Tevl, I-Ppol, I- Dirl, I-Hmul, I-HmuII, I-TevII, I-TevIII, F-Scel, F-Scell (HO), F-Suvl, F-Tevl and F-TevII or a derivative thereof. Further descriptions can be found, for example, in U.S. Pat. No. 6,238,924 and U.S. Patent Application No. US2008/0113437. These meganucleases are recombinant proteins with sequences that recognize sites (different from the wild meganuclease sites) and cause a double-stranded break in the DNA. Repair of the double-strand break by a homologous recombination mechanism leads to a mutation in the targeted site.

[00100] Further examples of methods and techniques that may be used in fish include those in Bedell V. M. et al. (2012) n vivo genome editing using a high-efficiency TALEN system” Nature 491 : 114-118; Ramirez C.L. et al. (2012)“Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects” Nucleic Acids Res. 40:5560- 5568; and Dow, L.E. (2015)“Modeling disease in vivo with CRISPR/Cas9” Trends Mol. Med. (l0):609-62l.

Non-Homologous End Repair [00101] In other embodiments, the fish of the invention may be made, for example, by non-homologous end joining (NHEJ) which tends to make insertions/deletions at multiple positions. Thus, the knockout may also be, for example produced by introducing a nuclease into the ova of fish to make breaks in the DNA and allow repair of the cleaved portions by non- homologous end joining. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced short palindromic repeats (CRISPRs) may be used to introduce targeted double stranded breaks in the chromosome of fish at the desired location. Repair by NHEJ does not require a repair template as in homology directed repair, but instead relies on the joining of overhangs in the DNA at the breaks. While often repaired without change, the method can lead to small insertions or deletions in the targeted area and may result in frameshift mutations. The frame shift mutation may then result in an effective knockout of the gene of interest. Screening for the desired mutations can reveal a knockout genotype on an allele of the fish. Heterozygous fish for a mstn-1 allele knockout may be crossed to produce homozygous fish carrying the same or different allelic mutations on each allele and produce a homozygous mstn-1 knockout phenotype. Examples of methods and techniques that may be used in fish include those in Amacher, S.L. (2008)“Emerging gene knockout technology in zebrafish: zinc-finger nucleases” Brief Funct. Genomic Proteomic 7(6):460-464; Valton J. el al. (2014)“Efficient strategies for TALEN-mediated genome editing in mammalian cell lines” Meth. San Diego Calif. 69: 151-170; and Varshney G.K. et al. (2015)“High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9” Genome Res. 25: 1030-1042. A review of such techniques may be found in Gaj, T. et al. (2013) “ZFN, TALEN and CRISPR/Cas-based methods for genome engineering” Trends Biotechnol. 3l(7):397-405.

[00102] Any other technique known in the art to knockout, truncate, disrupt, or edit the gene to create a heterozygous or homozygous fish with reduced or absent MSTN-l activity is within the scope of the invention.

[00103] The polynucleotide construct is inserted into fertilized tilapia ova by any means known such as viral transduction, lipofection, electroporation or direct injection (Sin F.Y.T. (1997)“Transgenic Fish” Rev. Fish Biol and Fisheries 7:417). For injection, the polynucleotide constructs are injected into the pronucleus (Rahman, M. A. and Maclean, N., (1992) Aquaculture 105:219-232). Rahman and MacLean found that a linear fragment increased the likelihood of integration and also decreased the occurrence of extrachromosomal copies of the gene.

[00104] Injection of fish fertilized eggs may be accomplished by positioning eggs such that the needle injects the micropyle to introduce the polynucleotides.

[00105] Once introduced into tilapia by microinjection, and mutagenesis is allowed to proceed and fish embryos are allowed to mature, fish are then screened for modified myostatin. Founder chimeric fish are crossed to obtain heterozygous Fl fish which can be further crossed to generate homozygous fish.

[00106] The invention will now be described by reference to the following examples which are meant to be illustrative of embodiments of the invention and are not to be construed as limiting the invention. The knockout tilapia of the invention may be used to generate tilapia with the additional trait described therein to produce a faster growing tilapia with the benefits of the benefits of the myostatin knockout.

EXAMPLES

Example 1

1. Methods and Materials

a. Mutations

[00107] Male and female Nile tilapia broodstock were kept in an Acrylic aquarium at 28°C and fed twice a day to satiation. Eggs were stripped from ripe females and fertilized with fresh milt. Mutation constructs were prepared to target mutations in the MSTN-l gene at Exon 1, 2, or 3. Mutations were made within a conserved region of 0. niloticus , I) rerio , 0. mykiss, and S. salar (sequence comparisons were made using Geneious). For Exon 3, mutations were made near the site of the natural myostatin mutations observed in MSTN in Belgian Blue (SEQ ID NO:3) and Piedmontese (SEQ ID NO:4) cattle.

b. Microinjections

[00108] Nile tilapia broodstock were obtained by the Institute of Marine & Environmental Technology from Dr. Thomas Kocher's lab at the ETniversity of Maryland (College Park). Broodstock were kept in an Acrylic aquarium at 28°C and fed twice a day to satiation. Eggs were stripped from ripe females and fertilized with fresh milt. Injected embryos were reared in the same water conditions as broodstock.

c. Screening for MSTN-1 Mutations

[00109] At l-m.p.f, the caudal fin of injected F0 fish was clipped, and genomic DNA was extracted with QuickExtract DNA Extraction Solution. A high-resolution melt assay (HRMA) was used to detect mutations. The assay was run on the QuantStudio 6 with MeltDoctor reagents (Applied Biosystems). The details on the HRMA primers from the Nile tilapia MSTN-l gene are shown in Table 1. Mutated chimeric fish were identified by comparing the melt curves to those of un-injected fish.

Table 1: HRMA primers

d. Heterozygous FI Mutants

[00110] Chimeric F0 fish with confirmed MSTN-l mutations were reared to maturity and crossed with WT fish to obtain heterozygous Fl mutants (Table 2). Fl fish were initially screened with the HRMA assay at l-m.p.f. At 3-m.p.f. , another caudal fin clip was taken from fish confirmed to be heterozygous MSTN-l mutants using the HRMA assay, and genomic DNA was extracted with QuickExtract DNA Extraction Solution. Standard PCR was performed on the genomic DNA, and next generation sequencing was performed by the Center for Computational & Integrative Biology DNA Core at Massachusetts General Hospital on the resulting PCR amplicons (primer details are shown in Table 3). Heterozygous Fl MSTN-l mutants with frameshift mutations (knockout alleles) were then reared to maturity.

Table 2: Heterozygous Fl Fish with Frameshift Mutations

Table 3: PCR primers for amplicons sequenced with NGS

f Small-Scale Growth Studies

[00111] Heterozygous Fl fish myostatin knockout alleles were crossed together in order to obtain mixed genotype batches containing homozygous F2 knockouts, heterozygous F2 mutants, and wild-type fish at Mendelian frequencies. Three different crosses (Batches A, B, and C) were enrolled in small-scale growth studies (Table 4). The three crosses were performed at different dates, but enrollment in the growth study occurred on the same date. Therefore, the three different Batches were of variable age and weight at the start of their respective growth studies.

Table 4: Mixed-genotype Batches enrolled in small-scale growth study

[00112] Before enrollment, Batches A, B, and C were reared in Acrylic aquariums at 28°C and fed twice a day to satiation. All three batches were then shipped to a different facility and transferred to recirculating aquaculture systems set at 29°C-30°C and with daily water renewal rates of 2.5-10% and tank water exchange rates greater than 2.5 -times per hour. Dissolved oxygen and temperature were monitored daily, and alkalinity, ammonia, nitrite, pH, and salinity were monitored three times per week. Fish were fed with a standard commercial tilapia diet via belt feeder once each hour for 14 hours per day. Feed rates were adjusted to maximize growth rates. When a Batch reached an average weight of 40-g, all fish in the Batch were tagged and caudal fin clips were taken for genotyping using the HRMA protocol described above. A subset of fish from each Batch was measured every week to determine the average tank body weight and monitor Batch growth status. Once tagged, all fish in a Batch had body weight and body length measured and condition index (K = 100 x Body Weight (g) / Body Length (cm) ^L 3) calculated at least once every 4 weeks. Batch A was harvested at an average body weight of 143- g, and all fish had final body weight, final body length, fillet weight, and viscera weight measured and final condition index, fillet yield (FY = Fillet Weight / Body Weight), and visceral-somatic index (VSI = Viscera Weight / Body Weight) calculated. Batch B and Batch C will be harvested at an average body weight of 550 g and 1,110 g, respectively.

2. Results

[00113] There were 26 crosses of chimeric F0 MSTN-l mutants with wild-type fish. A variety of different knockout alleles were identified in Exon 1 and Exon 3 (FIG. 2 A-D, FIG. 3).

[00114] Fl x Fl (Table 4) crosses were performed to generate the Batches of fish for small-scale growth studies. The data for the Batch A, B, and C growth studies are shown in FIGS. 4-10 (corresponding adjusted p-values are shown in Table 5). Between the three Batches, the average body weight for homozygous MSTN-l knockouts was 19-31% greater than wild- type fish (statistically significant only for Batches B and C). Notably, the average weight of the homozygous knockouts started to increase above that of the age-matched wild-type fish only after 50 g (FIG. 4). The average condition index, a measure of body thickness, for the knockouts was 49-67% greater than the wild-type controls (p<0.000l for all Batches). When filleted, Batch A homozygous knockouts had a 53% and 27% greater fillet weight (p=0.0002) and fillet yield (p<0.000l), respectively, than wild-type fish (FIG. 6).

Table 5: Adjusted p-values (p<0.05 bolded)

Example 2

Batch D and Batch F Studies

[00115] Heterozygous Fl fish myostatin knockout alleles were crossed together in order to obtain mixed-genotype batches containing homozygous F2 knockouts, heterozygous F2 mutants, and wild-type fish at Mendelian frequencies. Before enrollment in the growth studies, Batches D and F were reared in Acrylic aquariums at 28°C and fed twice a day at a low feed rate that maximized water quality. For each Batch, all fish were genotyped using the HRMA protocol described above and separated into three, genotype-specific tanks. The two genotyped batches were then shipped to the study facility (on separate dates) and transferred to recirculating aquaculture systems (RAS). For each Batch, fish were reared in three, genotype-specific tanks until enrollment in the studies.

[00116] During the studies, the RAS parameters were set at 29°C-30°C, daily water renewal rates of 2.5-10%, and tank water exchange rates greater than 2.5 -times per hour. Dissolved oxygen and temperature were monitored daily, and alkalinity, ammonia, nitrite, pH, and salinity were monitored three times per week. Fish were fed with a standard commercial tilapia diet via belt feeder once each hour for 14 hours per day. Within each study, the feed rate was constant between tanks, adjusted to optimize feed conversion rate (FCR), and reduced as the fish grew heavier. For all measurements and calculations, adjusted p-values were determined using Turkey’s multiple comparisons test in GraphPad Prism 7.01. a. Batch D Fillet Study Details

[00117] Fish were enrolled in the fillet study, and at enrollment, each genotype population was culled to minimize body weight variation. Eighty fish per genotype were split into 4 tanks per genotype (240 total fish, 12 total tanks). The tank weight (total body weight of all fish in a tank) was measured every week in order to monitor Batch growth status. The top 2 largest and smallest fish per tank (48 total fish, 16 fish per genotype, average weight 309-g) were harvested for fillet and loin analysis. All harvested Batch D fish were sexed and had final body weight, final body length, fillet weight, loin weight, viscera weight, liver weight, gonad weight measured. Fillet yield, loin yield (LY = Loin Weight / Body Weight), VSI, hepato-somatic index (HSI = Liver Weight / Body Weight), and gonado-somatic index (GSI = Gonad Weight / Body Weight) were calculated. b. Batch F Growth Study Details

[00118] In order to generate an all-male Batch, all Batch F fish were sex-reversed via methyltestosterone (MT) treatment. Fish were fed 60-mg MT per kg feed for 29 days from April 21, 2017 to May 19, 2017. After MT treatment, 19 of 22 fish (86%) were identified as male. Fish were shipped to the growth study facility on July 17, 2017. Fish were enrolled in the growth study on October 6, 2017. At enrollment, each genotype population was culled to minimize body weight variation, and 60 fish per genotype were split into 3 tanks per genotype (180 total fish, 9 total tanks). The tank weight (total body weight of all fish in a tank) was measured weekly in order to determine the average body weight and monitor Batch growth status. In order to determine FCR, the amount of feed provided to each tank was recorded. Specific growth rate (SGR = ((ln(Final Weight) - ln(Initial Weight)) x 100) / Study Duration in Days) and FCR (Total Feed Amount / (Final Weight - Initial Weight)) were calculated based on the initial and final weekly measurements. Altogether in three separate harvest events, n=36 Batch F fish of each genotype were harvested. All harvested Batch F fish were sexed and had final body weight, final body length, fillet weight, fillet length, fillet width, fillet thickness, viscera weight, and liver weight measured. For one harvest event (n=l2 fish of each genotype), additional measurements for loin weight, loin length, loin width and loin thickness were made. Fillet yield, loin yield, VSI and HSI were calculated. Details of Batch D and Batch F fish are shown in Table 6. c. Results

Table 6: Mixed-genotype Batches D and F enrolled in studies

* Sex-reversed. [00119] For every Batch D growth study tank, the top 2 largest fish at harvest were males, and the top 2 smallest fish were females. Homozygous knockout males were 25.1% (p<0.0l) heavier at harvest than wild-type males, and knockout females were 9.4% heavier than wild-type females (Table 7). The male homozygous knockout fillets weighed 96.7% (p<0.000l) more than wild-type fillets, with a 58.1% (pO.OOOl) increase in yield (FIG. 11). The male knockout loins weighed 163% (pO.OOOl) more than wild-type loins, with a 112% (pO.OOOl) increase in yield (FIG. 12) Similar trends were observed for the female fillets and loins. Body weight, weight gain and FCR for Batch D fish are shown in FIG. 13.

Table 7: Average Batch D harvest weights

[00120] For sex-reversed Batch F fish, the homozygous knockouts added 41.5% (p<0.005) more body weight, grew at a 14.2% faster specific growth rate, and had a 15.6% lower feed conversion rate than wild-type fish. Weekly body weight, weight gain, specific growth rate and FCR for Batch F fish from week 0 to week 6 are shown in FIG. 14. Body weight, weight gain, specific growth rate and FCR for Batch F fish from week 0 to week 19 are shown in FIG. 15.

[00121] Batch F homozygous knockouts harvested at when they reached a weight of about 850 g (n=32) weighed 61.9% (p<0.000l) more than wild-type, age-matched fish (n=32), with no significant difference in body length (FIG. 16). These homozygous knockout fillets (n=32) weighed 177% (p<0.000l) more than wild-type, age-matched fillets (n=32), with a 62.9% (p<0.000l) increase in yield, (p<0.000l) 22.8% increase in length, 44.0% (p<0.000l) increase in width and 111% (p<0.00l) increase in thickness (FIG. 17 and FIG. 18). The Batch F homozygous knockout loins (n=l2) weighed 302% (p<0.000l) more than wild-type, age- matched loins (n=l2), with a 120% (pO.OOOl) increase in yield, 24.8% (pO.OOOl) increase in length, 61% (r<0.0001) increase in width and 86.6% (pO.OOOl) increase in thickness (FIG. 17 and FIG. 18). Heterozygous, age-matched fish parameters are also shown in FIGS. 16-18.

[00122] A representative photograph of wildtype versus MSTN-l homozygous knockout fish is shown in FIG. 19.