Method for determining the sex of chicken embryos

Description

1 / 92 “METHOD FOR DETERMINING THE SEX OF CHICKEN EMBRYOS REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (EGG-P-010-PCT.xml; size: 59,207 bytes; and creation date: March 21, 2024) are incorporated into the present invention by reference in their entirety. CROSS-REFERENCE TO RELATED REQUEST

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 454,804, entitled “METHOD FOR GENDER DETERMINATION OF CHICKEN EMBRYOS”, filed March 27, 2023, the content of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[0003] The present invention relates to a transgenic chicken and methods for its use, such as for determining the sex of a chicken embryo in an unhatched egg. BACKGROUND OF THE INVENTION

[0004] The food industry uses broiler chickens for the production of chicken meat. These chickens are known to reach slaughter weight between four and six weeks of age. On the other hand, laying hens are raised primarily to concentrate all their energy on laying and therefore rarely reach a sufficient weight for meat production. Males of leaner breeds used in egg production cost more to feed and house than they would ever cost to be sold for meat, and are therefore economically less favorable to the industry. This is why billions of male chickens are slaughtered daily. Petition 870250101549, dated 06 / 11 / 2025, page 5 / 104 2 / 92 by asphyxiation or crushing, generating a global financial and ethical problem.

[0005] Sexing chicks is the method of distinguishing the sex of chickens and other offspring, usually by a trained person called a chick sexer or broiler sexer. Several methods are used to determine the sex of at least one day-old chick, including cloacal sexing, which is universal for all breeds but requires the work of a specialist, as well as feather sexing and color sexing, which are based on a sex-linked slow feathering gene and a sex-linked slow feathering gene, respectively. However, there is still a great need for in ovo sexing of an unhatched egg.

[0006] An automated system for determining the sex of a 14-day-old developing chick in ovo has been previously described. This system is specifically based on the substantial difference in feather coloration between males and females, which can be observed by candling a 14-day-old egg. In this system, the egg is candling with a halogen lamp. Although no negative effects have been observed in developing chicks, halogen lamps are known to produce significant amounts of heat that can negatively impact the embryo if the candling time is too long.

[0007] There is still a great need for new, robust, economical and accurate methods for in ovo sexing of embryos in unhatched eggs, specifically, that can be generally applied to any lineage of Petition 870250101549, dated 06 / 11 / 2025, p. 6 / 104 3 / 92 chicken and, even more, applicable in very early stages of embryonic development. SUMMARY OF THE INVENTION

[0008] According to one aspect, a transgenic female chicken is provided comprising a recombinant gender Z chromosome comprising a nucleic acid sequence of an exogenous reporter gene encoding a red fluorescent protein (RFP), at least one location on a gender Z chromosome of a female chicken, wherein at least one location is listed in Table 1.

[0009] In some embodiments, at least one location of a gender Z chromosome, as disclosed throughout the application, including Table 1, is in accordance with or based on the UCSC Genome Browser assembly ID: galGal6.

[0010] According to another aspect, a cell obtained or derived from the transgenic female chicken disclosed in the present invention is provided.

[0011] According to another aspect, a method is provided for determining the sex of a chicken embryo in an unhatched egg comprising the embryo within a structurally intact shell, the method comprising: (a) obtaining at least one unhatched egg comprising an embryo within a structurally intact shell, from the transgenic female chicken disclosed in the present invention; and (b) determining whether a red fluorescent signal is detected in the embryo residing in the unhatched egg, wherein the detection of the red fluorescent signal indicates the expression of RFP in the embryo within the structurally intact shell of the unhatched egg. Petition 870250101549, dated 06 / 11 / 2025, page 7 / 104 4 / 92 hatched and thus indicates the presence of the recombinant Z chromosome in the embryo, thus determining that the chicken embryo in the unhatched egg is a male embryo.

[0012] According to another aspect, a method is provided for producing a chicken comprising a recombinant gender Z chromosome, the method comprising: (a) obtaining at least one transgenic PGC comprising a recombinant Z chromosome comprising a nucleic acid sequence of an exogenous reporter gene encoding an RFP at least one location as listed in Table 1 on a chicken's gender Z chromosome; and, (b) transplanting the transgenic PGC into a receptive chicken embryo, thereby producing a chicken comprising the recombinant gender Z chromosome.

[0013] According to another aspect, a kit is provided comprising at least one first nucleic acid molecule encoding a guide RNA (gRNA), comprising the nucleotide sequence as presented in any of the SEQ ID Nos: 1 - 3, and 18 - 20.

[0014] In some modalities, at least one location is selected from the group consisting of: site 3, site 4, site 5, site 7, site 8, site 13, site 14, site 15, and any combination thereof.

[0015] In some modes, at least one location is listed in Table 3.

[0016] In some modalities, at least one location is selected from the group consisting of: site 4a, site 5a, site 7b, site 14a, and any combination thereof. Petition 870250101549, dated 06 / 11 / 2025, p. 8 / 104 5 / 92

[0017] In some modalities, at least one location is selected from the group consisting of: site 4a, site 13a, site 14a, and any combination thereof.

[0018] In some embodiments, the RFP is characterized by an excitation wavelength of 500 to 650 nm and an emission wavelength of 550 to 650 nm.

[0019] In some embodiments, the recombinant Z chromosome of gender comprises the nucleotide sequence presented in any of the SEQ ID Nos: 4 - 6.

[0020] In some forms, the cell is a primordial germ cell (PGC).

[0021] In some embodiments, the detection of the absence of red fluorescent signal in the resident embryo in the unhatched egg indicates that RFP is not expressed in the embryo and thus indicates the absence of recombinant Z chromosome in the embryo, thus determining that the chicken embryo in the unhatched egg is a female embryo.

[0022] In some embodiments, the method further comprises a step comprising subjecting the unhatched egg comprising the embryo within a structurally intact shell to a light source.

[0023] In some embodiments, the method additionally comprises a step prior to step (b) comprising isolating at least one female embryo from the transgenic female chicken.

[0024] In some embodiments, the kit additionally comprises at least one second nucleic acid molecule encoding any one of the following: a protein 9 Petition 870250101549, dated 06 / 11 / 2025, page 9 / 104 6 / 92 associated with clustered regularly interspersed short palindromic repeats (CRISPR), a PFR, and both.

[0025] In some embodiments, the kit additionally includes instructions for integrating at least the second nucleic acid molecule encoding RFP into at least one gender Z chromosome location of a female chicken, wherein the at least one location is listed in Table 1.

[0026] In some embodiments, the method additionally comprises a step prior to step (a), comprising integrating the nucleic acid sequence of an exogenous reporter gene encoding an RFP at least one location as listed in Table 1 into the gender Z chromosome of a PGC, thereby obtaining the transgenic PGC.

[0027] In some embodiments, the nucleic acid sequence of an exogenous reporter gene encoding an RFP is integrated into the gender Z chromosome location using a type II CRISPR system comprising Cas9 protein and a gRNA.

[0028] In some embodiments, the gRNA comprises the nucleotide sequence presented in any of the SEQ ID Nos: 1 - 3, and 18 - 20.

[0029] In some embodiments, the nucleic acid sequence of an exogenous reporter gene is integrated into site 4a, site 5a, or site 14a, using Cas9 protein and a gRNA comprising the nucleotide sequence as presented in SEQ ID NO: 1, SEQ ID NO: 20, or SEQ ID NO: 3, respectively.

[0030] Unless otherwise defined, all technical and / or scientific terms used in this invention have the same meaning commonly understood by Petition 870250101549, dated 06 / 11 / 2025, p. 10 / 104 7 / 92 a technician in the field to which the invention relates. Although methods and materials similar or equivalent to those described in this invention may be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including the definitions, shall prevail. Furthermore, the materials, methods, and examples are merely illustrative and are not intended to be necessarily limiting.

[0031] Additional embodiments and the full scope of applicability of the present invention will become apparent from the detailed description set forth below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are provided for illustrative purposes only, as various alterations and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0032] In addition to the exemplary aspects and modalities described above, additional aspects and modalities will become evident by reference to the study of the detailed description that follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 includes a gel electrophoresis image demonstrating cleavage products by a T7EI nuclease of double-stranded DNA (dsDNA) molecules from DF1 cells. Genomic DNA was edited by CRISPR-associated protein 9 (Cas9) and a guide RNA (gRNA) molecule selected from: gRNA6.1, gRNA7.2, and gRNA9.1, and amplified. Petition 870250101549, dated 06 / 11 / 2025, page 11 / 104 8 / 92 by PCR prior to cleavage by T7EI nuclease. +, with gRNA molecule; -, without gRNA molecule; m, marker.

[0034] Figure 2 includes a bar graph of indel tracking by decomposition analyses (TIDE), demonstrating the frequency of DNA insertions and deletions (indels) in DF1 genomic DNA treated with Cas9 / gRNA ribonucleoprotein (RNP) complexes. Indels were observed more frequently in the target genomic DNA of gRNA6.1, gRNA7.2, or gRNA9.1, compared to other gRNA molecules.

[0035] Figure 3 includes agarose gel electrophoresis photographs of assay T7, evaluating the efficiency of the designed guide RNAs (gRNAs). For sites 4a and 14a, the inventors used previously validated gRNAs (gRNA 6.1 and 9.1, respectively), known for their effectiveness in inducing double-strand breaks (DSB) at the target genomic loci. For the safe harbor loci (SHL) In Figures 7b, 8a, and 13a, the inventors designed and tested 2, 4, and 4 gRNAs, respectively. The symbols - and + indicate samples before and after treatment with the T7 endonuclease, respectively. The untransfected control represents PCR products obtained using equivalent T7 primers without RNP electroporation. In the assay, gRNAs 6.1, 6.1#2, and 7.2 were used as positive controls, representing RNP complexes with confirmed cleavage capacity, ensuring the reliability of the T7 assay. Additionally, NTC denotes the non-template control, serving to demonstrate the absence of contamination in the PCR assay.

[0036] Figures 4A-4F include fluorescent micrographs showing the genomic integration of the transgene into two SHLs. Fluorescence of DsRed (4A and 4D) and EGFP (4B and Petition 870250101549, dated 06 / 11 / 2025, page 12 / 104 9 / 92 Figure 4E) observed 2 to 3 days after transfection of PGC LohmannLSL targeting two different genomic SHLs, representing the expression of the HDR and CRISPR / Cas9 plasmids of DsRed, respectively. The blended field (4C and 4F) demonstrates the colocalization of red and green fluorescence in some of the cells.

[0037] Figures 5A-5B include fluorescent micrographs showing stable primordial germ cell (PGC) cultures expressing DsRed directed to two SHLs: 4a (5A) and 13a (5B).

[0038] Figures 6A-6B include agarose gel electrophoresis photographs of PCR assays that confirm the precise integration of the DsRed encoding gene into SHL 4a (6A) and 13a (6B), with the expected product sizes. For SHL 13a, two sets of primers (#1 and #2) were used for each homology arm.

[0039] Figures 7A-7C include fluorescent micrographs showing in ovo expression of DsRed in PGC containing the DsRed encoding gene embedded in an SHL in the genome. Non-fertile eggs were injected with: unedited control PGC (7B) or edited PGC containing the DsRed encoding gene in an SHL in the genome in an amount equivalent to the number of cells on day 3 of the embryo (7C), and subsequently scanned using an electro-optical scanner. A distinct fluorescent focus was detectable only in the egg injected with DsRed+ cells, as indicated by the arrow (7C). (7A) An untreated control egg. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention, in some embodiments, provides a transgenic female chicken comprising a Petition 870250101549, dated 06 / 11 / 2025, page 13 / 104 10 / 92 recombinant gender Z chromosome comprising a nucleic acid sequence of an exogenous reporter gene encoding a red fluorescent protein (RFP). The present invention is further directed to a method for determining the sex of a chicken embryo in an unhatched egg comprising the embryo within a structurally intact shell. Also provided in the present invention are a kit comprising at least one nucleic acid molecule encoding a guide RNA (gRNA), comprising a nucleotide sequence as presented in SEQ ID Nos: 1 - 3, and 18 - 20, and a method for producing a chicken comprising a recombinant gender Z chromosome. Genetically modified chicken, its cell, and the method for producing it.

[0041] According to another aspect, a transgenic female chicken is provided comprising a recombinant gender Z chromosome comprising a nucleic acid sequence of an exogenous reporter gene encoding a red fluorescent protein (RFP), at least one location on the gender Z chromosome, wherein at least one location is listed in Table 1.

[0042] According to another aspect, a transgenic chicken cell is provided comprising a recombinant Z chromosome of genus comprising a nucleic acid sequence of an exogenous reporter gene encoding a red fluorescent protein (RFP), at least one location on the Z chromosome of genus, wherein at least one location is listed in Table 1. Petition 870250101549, dated 06 / 11 / 2025, page 14 / 104 11 / 92

[0043] In some embodiments, the transgenic chicken cell is a stable cell. In some embodiments, the transgenic chicken cell is genomically stable.

[0044] In some embodiments, the stability, genomic stability, fitness, survival, viability, or any combination thereof, of the transgenic cell of the invention, is essentially similar to that of a control. In some embodiments, a control is or comprises a control chicken cell.

[0045] In some embodiments, a control chicken cell comprises a wild-type chicken cell, a cell derived from or from a transgenic chicken cell gene or reference, or both.

[0046] In some embodiments, “essentially similar” refers to being at least 80%, 90%, 95%, 97%, or 99% similar to a control cell as described in the present invention, or any value and any value and range therebetween. In some embodiments, “essentially similar” refers to being 80 to 100%, 85 to 100%, 90 to 100%, 95 to 100%, or 97 to 100% similar to a control cell as described in the present invention. Each possibility represents a distinct embodiment of the invention.

[0047] In some embodiments, the transgenic chicken cell is characterized by RFP expression being comparable to a control, proliferation rate being comparable to a control, or both.

[0048] In some embodiments, the transgenic chicken cell is characterized by stable RFP expression, stable proliferation rate, or both. In some embodiments, stable is comparable to a control. Petition 870250101549, dated 06 / 11 / 2025, page 15 / 104 12 / 92

[0049] In some embodiments, stable means stable over time. In some embodiments, stable means for a period of time. In some embodiments, stable means for at least 1 pass, 2 passes, 4 passes, 5 passes, or any value and range between them. Each possibility represents a distinct embodiment of the invention.

[0050] In some embodiments, stable is for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 4 months, 6 months, or any value and range between them. In some embodiments, stable is for 1 to 3 weeks, 2 to 6 weeks, 3 to 7 weeks, 1 to 3 months, 2 to 6 months, or 4 to 6 months. Each possibility represents a separate embodiment of the invention.

[0051] The terms “comparable” and “essentially similar” are used interchangeably in the present invention.

[0052] In some modalities, essentially similar includes or is 100% similar, for example, 'identical'.

[0053] As used in the present invention, the terms stable or genomically stable refer to a cell that does not lose fitness, survivability, viability, activity or the like, due to the presence of the exogenous reporter gene nucleic acid sequence encoding a red fluorescent protein (RFP), in at least one location on the cell's gender Z chromosome, wherein the at least one location is listed in Table 1.

[0054] In some embodiments, the transgenic chicken cell of the invention is characterized by consistently stable RFP expression. In some embodiments, the transgenic chicken cell of the invention is characterized by Petition 870250101549, dated 06 / 11 / 2025, page 16 / 104 13 / 92 sustained proliferation. In some embodiments, the transgenic chicken cell of the invention is characterized by consistently stable RFP expression and sustained proliferation.

[0055] Methods for determining RFP expression over time (e.g., consistent and stable RFP expression), as well as cell proliferation over time (e.g., sustained proliferation), are common and would be self-evident to someone with common knowledge of the art, as exemplified in the present invention.

[0056] The term “transgenic chicken”, as used in the present invention, refers to a chicken (Gallus gallus) that is genetically modified so that the chicken's genome comprises an exogenous DNA sequence incorporated into it. In some embodiments, the exogenous DNA sequence is integrated into a gender chromosome of a germ cell of the transgenic chicken. As a result of this integration, the exogenous sequence can be transmitted through the germ cells to the offspring of a transgenic chicken. The transgenic chicken, including its progeny, also possesses the exogenous reporter gene integrated into the gender chromosomes of the somatic cells. As used in the present invention, the terms “location,” “locus,” and “genomic site” are interchangeable and refer to a site within a chromosome where the exogenous reporter gene can be integrated. A genomic site can refer to a location between two consecutive nucleotides in genomic DNA, as well as to a range of consecutive nucleotides comprising up to 6,000,000 nucleotides. The term “Z gender chromosome,” as used in the present invention, Petition 870250101549, dated 06 / 11 / 2025, page 17 / 104 14 / 92 invention, refers to the male sex chromosome in chickens. As used in the present invention, the term recombinant Z-gender chromosome refers to a Z-gender chromosome from a chicken that has been genetically modified to include a nucleic acid from an exogenous reporter gene encoding RFP. In some embodiments, the genetic modification is performed by a gene editing tool or system.

[0057] In some embodiments, at least one location is selected from: comprising nucleotide number 1,550,000 to nucleotide number 1,730,000 of a chicken Z chromosome, site 2, comprising nucleotide number 4,075,000 to nucleotide number 6,150,000 of a chicken Z chromosome, site 3, comprising nucleotide number 17,100,000 to nucleotide number 17,450,000, site 4, comprising nucleotide number 17,550,000 to nucleotide number 18,050,000, site 5, comprising nucleotide number 19,500,000 to nucleotide number 20,000,000, site 6, comprising nucleotide number 26,000,000 to nucleotide number 26,400,000, site 7, comprising nucleotide number 30,000,000 to nucleotide number 30,800,000, site 8, comprising nucleotide number 36,000,000 to nucleotide number 36,600,000, site 9, comprising nucleotide number 38,500,000 to nucleotide number 39,200.000, site 10, comprising nucleotide number 48,300,000 to nucleotide number 50,000,000, site 11, comprising nucleotide number 50,900,000 to nucleotide number 51,400,000, site 12, comprising nucleotide number 51,425,000 to nucleotide number. Petition 870250101549, dated 06 / 11 / 2025, page 18 / 104 15 / 92 51,625,000, site 13, comprising nucleotide number 58,500,000 to nucleotide number 59,700,000, site 14, comprising nucleotide number 61,400,000 to nucleotide number 62,400,000, site 15, comprising nucleotide number 63,150,000 to nucleotide number 63,550,000, site 16, comprising nucleotide number 64,350,000 to nucleotide number 65,250,000, site 17, comprising nucleotide number 69,700,000 to nucleotide number 70,400,000, site 18, comprising the number of nucleotides from 73,500,000 to number of nucleotides 78,800,000, or any combination thereof.

[0058] In some embodiments, at least one location comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 locations selected from sites Nos. 1-18, as disclosed in the present invention, or any combination thereof. Each possibility represents a distinct embodiment of the invention. In some embodiments, at least one location is a location selected from sites Nos. 1-18, as disclosed in the present invention.

[0059] In some embodiments, at least one location is selected from: site 3, site 4, site 5, site 7, site 8, site 13, site 14, site 15, or any combination thereof. In some embodiments, at least one location comprises 1, 2, 3, 4, 5, 6, 7, or 8 locations selected from sites Nos. 3, 4, 5, 7, 8, 13, 14, and 15, as disclosed in this invention, or any combination thereof. Each possibility represents a distinct embodiment of the invention. In some embodiments, at least one location is a location Petition 870250101549, dated 06 / 11 / 2025, p. 19 / 104 16 / 92 selected from sites Nos. 3, 4, 5, 7, 8, 13, 14 and 15, as disclosed in this invention.

[0060] In some embodiments, at least one location is listed in Table 3. In some embodiments, site 3 comprises nucleotide number 17,141,000 to nucleotide number 17,406,000 (site 3a) of a chicken Z chromosome, site 4 comprises nucleotide number 17,735,000 to nucleotide number 18,020,000 (site 4a), site 5 comprises nucleotide number 19,560,000 to nucleotide number 19,995,000 (site 5a), site 7 comprises nucleotide number 30,060,000 to nucleotide number 30,270,000 (site 7a), or nucleotide number 30,465,000 to nucleotide number 30,750,000 (site 7b), site 8 comprises nucleotide number 36,215,000 to nucleotide number 36,545,000 (site 8a), site 13 comprises nucleotide number 59,100,000 to nucleotide number 59,430,000 (site 13a), site 14 comprises nucleotide number 61,530,000 to nucleotide number 61,865,000 (site 14a), or between nucleotide number 61,875,000 and nucleotide number 62,315.000 (site 14b), and site 15 comprises nucleotide number 63,210,000 through nucleotide number 63,440,000 (site 15a).

[0061] In some embodiments, at least one location comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 locations selected from Sites Nos. 3a, 4a, 5a, 7a, 7b, 8a, 13a, 14a, 14b, and 15a, as disclosed in this invention, or any combination thereof. Each embodiment represents a distinct embodiment of the invention. In some embodiments, at least one location is a location selected from Sites Nos. 3a, Petition 870250101549, dated 06 / 11 / 2025, p. 20 / 104 17 / 92 4a, 5a, 7a, 7b, 8a, 13a, 14a, 14b and 15a, as disclosed in this invention.

[0062] In some embodiments, at least one location is selected from: site 4a, site 5a, site 7b, site 13a, site 14a, or any combination thereof. In some embodiments, at least one location comprises 1, 2, 3, or 4 locations selected from sites Nos. 4a, 5a, 7b, 13a, and 14a, as disclosed in this invention, or any combination thereof. Each possibility represents a distinct embodiment of the invention. In some embodiments, at least one location is a location selected from sites Nos. 4a, 5a, 7b, 13a, and 14a, as disclosed in this invention.

[0063] In some embodiments, at least one location is selected from: site 4a, site 5a, site 13a, site 14a, or any combination thereof. In some embodiments, at least one location comprises 1, 2, or 3 locations selected from sites Nos. 4a, 5a, 13a, and 14a, as disclosed in this invention, or any combination thereof. Each possibility represents a distinct embodiment of the invention. In some embodiments, at least one location is a location selected from sites Nos. 4a, 5a, 13a, and 14a, as disclosed in this invention.

[0064] As used in the present invention, the term “reporter gene” encompasses a gene encoding a polypeptide, the expression of which can be detected in a variety of known assays and wherein the level of the detected signal indicates the presence of the reported gene. Petition 870250101549, dated 06 / 11 / 2025, p. 21 / 104 18 / 92

[0065] As used in the present invention, the term “red fluorescent protein” or “RFP” refers to a fluorophore that emits orange, red, and far-red fluorescence that has been isolated from anthozoans or anemones, or any variant thereof. In some embodiments, the RFP comprises the DsRed protein. In some embodiments, the DsRed protein is isolated from Discosoma striata. In some embodiments, the RFP comprises the Kaede protein, isolated from Trachyphyllia geoffroyi. In some embodiments, the RFP comprises a variant of the RFP. In some embodiments, the RFP variant disclosed in this invention exhibits orange, red, far-red fluorescence, or any combination thereof. In some embodiments, the RFP variant comprises a monomeric variant. In some embodiments, the RFP variant comprises a reduced time between protein synthesis and fluorescence expression (e.g., maturation time), compared to DsRed.Variants of RFP are known in the art. Non-limiting examples include mFruits (mCherry, mOrange, mRaspberry), mKO, TagRFP, mKate, mRuby, FusionRed, mScarlet, and DsRed-Express.

[0066] In some forms, RFP includes or is DsRed-Express.

[0067] In some embodiments, an RFP or an analogue thereof is characterized by or has excitation at a wavelength of 500 to 650 nm. In some embodiments, an RFP or an analogue thereof is characterized by or has excitation at a wavelength of 510 - 650, 520 - 650, 530 - 650, 540 - 650, 550 - 650, 500 - 600, 510 - 600, 520 - 600, 530 - 600, 540 - 600, 550 - 600, 500 - 590, 510 Petition 870250101549, dated 06 / 11 / 2025, p. 22 / 104 19 / 92 590, 520 - 590, 530 - 590, 540 - 590, 550 - 590, 500 - 580, 510 - 580, 520 - 580, 530 - 580, 540 - 580, 550 - R$ 510 - 570, 520 - 570, 530 - 570, 540 - 570, 550 570, 500 - 560, 510 - 560, 520 - 560, 530 - 560, 540 - 560, or 550 - 560 nm. Each possibility represents a distinct embodiment of the invention.

[0068] In some embodiments, an RFP or an analogue thereof is characterized by or has emission at a wavelength of 550 to 650 nm. In some embodiments, an RFP or analogue thereof is characterized by or has excitation at a wavelength of 500 to 650, 510 to 650, 520 to 650, 530 to 650, 540 to 650, 550 to 650, 560 to 650, 570 to 650, 580 to 650, 500 to 640, 510 to 640, 520 to 640, 530 to 640, 540 to 640, 550 to 640, 560 to 640, 570 to 640, 580 to 640, 500 to 630, 510 to 630, 520 to 630, 530 to 630. 540 - 630, 550 - 630, 560 - 630, 570 - 630, 580 - 630, 500 - 620, 510 620, 520 - 620, 530 - 620, 540 - 620, 550 - R. 580 610, 500 - 600, 510 - 600, 520 - 600, 530 - 600, 540 - 600, 550 - 600, 560 - 600, 570 - 600, 580 - 600, 500 - R$ 590, 520 - 590, 530 - 590, 540 - 590, 550 - 590, 560 590, 570 - 590, or 580 - 590 nm.Each possibility represents a distinct embodiment of the invention.

[0069] Additional examples for RFP analogues are disclosed in WO2017094015A1, which is incorporated into the present invention by reference in its entirety.

[0070] In one embodiment, the maximum RFP excitation is about 554 nm. In some embodiments, the maximum RFP emission is about 586 nm. Petition 870250101549, dated 06 / 11 / 2025, p. 23 / 104 20 / 92

[0071] The term “approximately” as used in the present invention indicates values ​​that may differ by up to: 1%, 5%, 10%, 15%, or 20% more or less than the referenced value, the deviation range including whole numbers and, if applicable, non-whole numbers, constituting a continuous range. As used in the present invention, the term “approximately” refers to ± 10%.

[0072] In some embodiments, the RFP disclosed in this invention comprises the amino acid sequence: MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGRPYEGTQTAKLKVT KGGPLPFAWDILSPQFQYGSKVYVKHPADIDYKKLSFPEGFKWERVMNFEDGGVVTVT QDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERGWKLGLKLKLKLKVT KDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHHLFL (SEQ ID NO: 7), or an analogue or variant thereof.

[0073] The terms “variant” and “analog” are used interchangeably in the present invention. An RFP polypeptide analog includes any polypeptide that is similar, but not identical, to a polypeptide comprising the amino acid sequence presented in SEQ ID NO: 7, provided that it has or maintains at least 70%, 80%, 90%, or 95%, or any value and range therebetween, of the maximum excitation and / or maximum RFP emission, as disclosed in the present invention. Each possibility represents a separate embodiment of the invention. In some embodiments, an RFP polypeptide analog maintains 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, or 99% to 100% of the maximum excitation and / or maximum RFP emission, as disclosed in the present invention. Each possibility represents a separate embodiment of the invention. It should be understood that an RFP analog refers to any fluorescent protein. Petition 870250101549, dated 06 / 11 / 2025, p. 24 / 104 21 / 92 characterized by being capable of excitation at wavelengths from 500 to 650 nm and emission at wavelengths from 550 to 650 nm.

[0074] In some embodiments, the reporter gene encoding RFP comprises a codon sequence optimized for expression in a chicken cell. As used in the present invention, the term “optimized codon sequence” describes a sequence that encodes amino acids identical to those encoded by a non-optimized codon sequence (synonymous codon), but at least one of the following: translation rate of the optimized codon sequence, amount of protein product, duration of protein structure stability, or any combination thereof, is increased compared with the non-optimized codon. A person skilled in the art will know how to optimize a codon sequence for its expression in the desired cell using a codon optimization genetic engineering tool, comprising, among others, algorithms that analyze codon optimization based on codon frequencies in the desired cell / species.In some modalities, the increase in one of the following: translation rate, amount of protein product, and duration of structure stability is at least 30%.

[0075] In some embodiments, the reporter gene encoding RFP comprises the nucleic acid sequence: ATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGG CTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGG GCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGAC ATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCGCCGACAT CCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACT TCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCTCCTTC Petition 870250101549, dated 06 / 11 / 2025, p. 25 / 104 22 / 92 ATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTAATGCAGAA GAAGACTATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGA AGGGCGAGATCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTC AAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTGGACTC CAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCG CCGAGGGCCGCCACCACCTGTTCCTGTAG (SEQ ID NO: 8).

[0076] In some embodiments, the reporter gene encoding RFP comprises the nucleic acid sequence as presented in SEQ ID NO: 8, or an analog thereof, having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identity or homology with the same, or any value and range between them. Each possibility represents a distinct embodiment of the invention. In some embodiments, an RFP analog, comprising 50 to 100%, 60 to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 99% to 100% identity or homology with a nucleic acid sequence as presented in SEQ ID NO: 8. Each possibility represents a distinct embodiment of the invention.

[0077] In some embodiments, an RFP analog comprises a protein translated by a nucleic acid molecule comprising a nucleotide sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identity or homology with a nucleotide sequence as presented in SEQ ID NO: 8, or any value and range between them. Each possibility represents a distinct embodiment of the invention. In some embodiments, an RFP analog comprises a protein translated by a nucleic acid molecule comprising a nucleotide sequence having 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 99% to 100% of Petition 870250101549, dated 06 / 11 / 2025, page 26 / 104 23 / 92 identity or homology with a nucleotide sequence as presented in SEQ ID NO: 8. Each possibility represents a distinct embodiment of the invention.

[0078] In some embodiments, the reporter gene encoding RFP is integrated into the genomic site on the Z chromosome using a programmable engineered nuclease (PEN).

[0079] The term “programmable engineered nuclease (PEN) as used in the present invention, refers to a synthetic enzyme that cuts specific DNA sequences, derived from natural nucleases involved in DNA repair of double-strand DNA lesions, and allows direct genome editing.

[0080] In some embodiments, PEN used by the methods of the invention may be any of the grouped class I or class II regularly interspaced short palindromic repeat (CRISPR) systems.

[0081] As used in the present invention, CRISPR or “CRISPR arrays,” also known as SPIDRs (Spacer Interspersed Direct Repeats), constitute a family of recently described DNA loci, generally specific to a particular bacterial species. The CRISPR array is a distinct class of short sequence interspersed repeats (SSRs) that were first recognized in E. coli. In subsequent years, similar CRISPR arrays were found in Mycobacterium tuberculosis, Haloferax mediterranei, Methanocaldococcus jannaschii, Thermotoga maritima, and other bacteria and archaea. It should be understood that the invention contemplates the use of any of the known CRISPR systems, particularly the CRISPR systems disclosed in the present invention. Petition 870250101549, dated 06 / 11 / 2025, p. 27 / 104 The CRISPR-Cas system targets DNA molecules based on short sequences of homologous DNA, called spacers, that exist between the repeats. These spacers guide CRISPR-associated proteins (Cas) to corresponding (and / or complementary) sequences within the foreign DNA, called protospacers, which are subsequently cleaved. Spacers can be rationally designed to target any DNA sequence. Furthermore, this recognition element can be designed separately to recognize and target any desired target. Regarding CRISPR systems, as will be recognized by experts in the field, the structure of a natural CRISPR locus includes a series of short, repetitive sequences, generally called repeats. The repeats occur in clusters and are usually regularly spaced by intervening unique sequences, called spacers.Typically, CRISPR repeats range from about 24 to 47 base pairs (bp) in length and are partially palindromic. Spacers are located between two repeats, and typically each spacer has unique sequences with lengths between 20 or less and 72 or more bp. In some embodiments, the CRISPR spacers used in the sequence encoding at least one gRNA of the methods and kits of the invention comprise between 10 and 75 nucleotides (nt) each. In some embodiments, the gRNA comprises at least: 10, 11, 12, 13, ... 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or any value and range in between. Each Petition 870250101549, dated 06 / 11 / 2025, page 28 / 104 Possibility 25 / 92 represents a distinct embodiment of the invention. In some embodiments, the gRNA comprises 70 to 150 nt. In some embodiments, the spacers comprise 20 to 35 nucleotides. In addition to at least one repeat and at least one spacer, a CRISPR locus also includes a leader sequence and, optionally, a sequence encoding at least one tracrRNA. The leader sequence is typically an AT-rich sequence of up to 550 bp directly adjacent to the 5' end of the first repeat.

[0082] In some embodiments, the PEN used by the methods of the invention may be a Class 2 CRISPR system. In some additional particular embodiments, such a Class 2 system may be a Type II CRISPR system. In some embodiments, the PEN comprises a Type II CRISPR system.

[0083] The term “CRISPR type II system” refers to a bacterial immune system that has been modified for genomic engineering. However, it should be considered that other genomic engineering approaches, such as zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), which rely on the use of customizable DNA-binding protein nucleases, requiring the design and generation of a specific nuclease pair for each genomic target, may also be applicable in the present invention. CRISPR-Cas systems are divided into two classes. Class 1 systems utilize a complex of multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems utilize a single large Cas protein for the same purpose. More specifically, class 1 can be divided into types I, Petition 870250101549, dated 06 / 11 / 2025, page 29 / 104 26 / 92 Class III and IV, and class 2 can be divided into types II, V, and VI.

[0084] CRISPR-Cas type II systems include the 'HNH' type system (similar to Streptococcus; also known as the Nmeni subtype, for Neisseria meningitidis serogroup A str. Z2491, or CASS4), in which Cas9 is sufficient to generate crRNA and cleave the target DNA, in addition to the ubiquitous Cas1 and Cas2. Cas9 contains at least two nuclease domains, a RuvC-like nuclease domain near the amino terminus and the HNH (or McrA-like) nuclease domain in the middle of the protein, but the function of these domains still needs to be elucidated. However, as the HNH nuclease domain is abundant in restriction enzymes and possesses endonuclease activity responsible for cleaving the target.

[0085] Type II systems cleave precrRNA via an unusual mechanism involving the formation of a duplex between a tracrRNA and part of the repeat in the precrRNA; the first cleavage in the precrRNA processing pathway subsequently occurs in this repeat region. Furthermore, it should be noted that the Type II system comprises at least one of the Cas9, Cas1, Cas2 csn2, and Cas4 genes. It should be considered that any Type II CRISPR-Cas system may be applicable in the present invention, specifically, any of the Type II-A or B systems.

[0086] In some embodiments, at least one Cas gene used in the methods and kits of the invention may be at least one Cas gene of the CRISPR type II system (type II-A or type II-B). In some embodiments, at least one Cas gene of the CRISPR type II system used by the methods and kits of the invention is the Cas9 gene. It should be considered that such a system Petition 870250101549, dated 06 / 11 / 2025, p. 30 / 104 27 / 92 may additionally comprise at least one of the following genes: Casl, Cas2, cn2, and Cas4.

[0087] In some embodiments, a Cas protein consists of or comprises a Cas9 protein. The cleavage of double-stranded DNA (dsDNA) by Cas9 is a hallmark of type II CRISPR-Cas immune systems. The CRISPR-associated Cas9 protein is an RNA-guided DNA endonuclease that utilizes RNA:DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage, creating the double-strand brakes (DSBs) necessary for HDR, resulting in the integration of the reporter gene into the specific target sequence, for example, a specific target within the avian Z chromosome. The target DNA sequences are specified by the CRISPR array, which is a series of spacers of approximately 30 to 40 bp separated by short palindromic repeats.The array is transcribed as a precrRNA and processed into shorter crRNAs that associate with the Cas protein complex to target complementary DNA sequences known as protospacers. These protospacer targets must also have an additional neighboring sequence known as a protospacer-adjacent motif (PAM), necessary for target recognition. After binding, a Cas protein complex acts as a DNA endonuclease to cleave both strands in the target, and subsequent DNA degradation occurs via exonuclease activity.

[0088] The CRISPR type II system, as used in the present invention, requires the inclusion of two essential components: a guide RNA (gRNA) and a non-iononuclease. Petition 870250101549, dated 06 / 11 / 2025, page 31 / 104 28 / 92 specific CRISPR-associated (Cas9). The gRNA is a short synthetic RNA composed of a backbone sequence necessary for binding to Cas9 and a spacer or target sequence approximately 20 nucleotides long, which defines the genomic target to be modified. Thus, it is possible to alter the Cas9 genomic target simply by changing the targeting sequence present in the gRNA. Guide RNA (gRNA), as used in the present invention, refers to a synthetic fusion of endogenous bacterial crRNA and tracrRNA, providing targeting specificity and backbone / binding capacity for the Cas9 nuclease. Also known as single guide RNA or sgRNA.CRISPR was originally employed for knocking out target genes in various cell types and organisms, but modifications to the Cas9 enzyme have broadened CRISPR's application to knocking in target genes, selectively activating or repressing target genes, purifying specific regions of DNA, and even imaging DNA in living cells using fluorescence microscopy. Furthermore, the ease of generating gRNAs makes CRISPR one of the most scalable genome editing technologies and it has recently been used for complete genome screenings.

[0089] In some embodiments, the type II CRISPR system used for the method disclosed in the present invention comprises CRISPR-associated endonuclease 9 (Cas9) and a gRNA. In some embodiments, the gRNA disclosed in the present invention comprises a nucleotide sequence selected from: ACATGCAATACACTGAACTG (SEQ ID NO: 1), ACTGTTAACAAGGTTGGTTG (SEQ ID NO: 2), CTTGTAGGGCTTGATTACTG (SEQ ID NO: 3), TCTTGCTTTCCACTTTCCAT Petition 870250101549, dated 06 / 11 / 2025, p. 32 / 104 29 / 92 (SEQ ID NO: 18), AAGTGTGGAACAAACTGCTG (SEQ ID NO: 19) GATGGGCTCCACAAGGAACT (SEQ ID NO: 20), or any combination thereof. In some embodiments, the gRNA disclosed in the present invention comprises a nucleotide sequence as shown in SEQ ID NO: 1. In some embodiments, the gRNA comprises a nucleotide sequence as shown in SEQ ID NO: 2. In some embodiments, the gRNA comprises a nucleotide sequence as shown in SEQ ID NO: 3. In some embodiments, the gRNA comprises a nucleotide sequence as shown in SEQ ID NO: 18. In some embodiments, the gRNA comprises a nucleotide sequence as shown in SEQ ID NO: 19. In some embodiments, the gRNA comprises a nucleotide sequence as shown in SEQ ID NO: 20.

[0090] In some embodiments, gRNA comprising a nucleotide sequence as presented in SEQ ID NO: It is used for the integration of the exogenous reporter gene at site 4a. In some embodiments, gRNA comprising a nucleotide sequence as presented in SEQ ID NO: 20 is used for the integration of the exogenous reporter gene at site 13a. In some embodiments, gRNA comprising a nucleotide sequence as presented in SEQ ID NO: 3 is used for the integration of the exogenous reporter gene at site 14a.

[0091] In some embodiments, the recombinant Z chromosome of gender comprises the nucleotide sequence: GGCCTATACAGACAAATCTTTACTAAATGGAGAATCCTGACATTTTGTC CATTTCTCTCTATACCACCAACTCTCATCTTGGTTACATTGCCTGTCAGTTGAACAGGC AAGATCTCCATTCCCAAAACACCACACTTATCTTGAATTGAGCCATCAAGAGGTATTTC TCTGATACACCATTATCTAAAACCCAGTGTTATTTTTCAGTGAGGGTAGATTCCCTTTA Petition 870250101549, dated 06 / 11 / 2025, p. 33 / 104 30 / 92 CTGCTTTTTCTCCTCATTTCCAGAATTCCGTTACATAACTTACGGTAAATGGCCCGCCT GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACTTT CCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAG TGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG CATTGTGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATT AGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCT CCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCG ATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCG GGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGT TTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGG GCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCG CCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTT CTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGT GGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGG CGCCACCATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCA TGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCC TACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGC CTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCG CCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTG ATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGG CTCCTTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTAA TGCAGAAGAAGACTATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGC GTGCTGAAGGGCGAGATCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACG TGGACTCCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGTAC GAGCGCGCCGAGGGCCGCCACCACCTGTTCCTGTAGCGGCCGCGACTCTAGATCATAAT CAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCC TGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTAT AATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACT GCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTGGATCCATAAC Petição 870250101549, de 06 / 11 / 2025, pág. 34 / 104 31 / 92 GGCATTCAGTACTTGTCATCACTAATTAGTGTATTCAATCTACCCTCTATTTCTAGGAC AACATGTTTTAATAATTCACCATAAATTAGTTCTATGAGCATTTTGATCTAGTGGTTGT TGTAGATCACAGGATATGAATAGATTGTGTGTATGTGTAAACCAGGAGTGTCTTGACAA AAGGACACAGTGCTGGTAAGACCAGCAGAAGAGGGTCCCCTGTGGCAAAAATGTTTTAC TGACCTTTG (SEQ ID NO: 4).

[0092] In some embodiments, the recombinant Z chromosome of gender comprising the exogenous reporter gene at site 4a comprises the nucleotide sequence as presented in SEQ ID NO: 4.

[0093] In some embodiments, the recombinant gender Z chromosome comprises the nucleotide sequence as presented in SEQ ID NO: 4, or an analogue thereof, having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identity or homology thereto, or any value and range therebetween. Each possibility represents a distinct embodiment of the invention. In some embodiments, an analogue of the nucleotide sequence as presented in SEQ ID NO: 4, comprises 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 99% to 100% identity or homology with a nucleic acid sequence as presented in SEQ ID NO: 4. Each possibility represents a distinct embodiment of the invention.

[0094] In some embodiments, the recombinant Z chromosome of gender comprises the nucleotide sequence: AATACACAGCACTGTATCTTTTATGGCAATCTATTTTCGATTGTCTCAT GTGCAGAACAGTTACCAGGATTGCAACAACGGATTGAATTTATCCGAAAAGAAGTTTTG TGTCCTGCTTTGTGATAGCTGAGAAAGAAAGGCAGTGATGCTTAAAAAGCAGTCAGTGA CCTAATCACCTACTGTCAGGTGTACTATGAATACATACAGTAGAGCCAGTAACACAGTT TGACAGCATTTTCATTAGATGTTTGAATTCCGTTACATAACTTACGGTAAATGGCCCGC Petition 870250101549, dated 06 / 11 / 2025, page 35 / 104 32 / 92 CTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACT TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCA AGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTGTGCCCAGTACATGACCTTATGGACTTTCCTACTTGGCAGTACATCTACGTA TTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCAT CTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAG CGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGG CGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAA GTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGC GGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGC CGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCC TTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTG GTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTT GGCGCCACCATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCG CATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCC CCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTC GCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCC CGCCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCG TGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGAC GGCTCCTTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGT AATGCAGAAGAAGACTATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACG GCGTGCTGAAGGGCGAGATCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTG GTGGAGTTCAAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTA CGTGGACTCCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGT ACGAGCGCGCCGAGGGCCGCCACCACCTGTTCCTGTAGCGGCCGCGACTCTAGATCATA ATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCC CCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTT ATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCA CTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTGGATCCCAGAACAAAATAGTATTTTTGTTCACAACTGGGAGTGAAATCTGATTTCAAACCACTAAAAA Petition 870250101549, dated 06 / 11 / 2025, p. 36 / 104 33 / 92 GAATAGTGGAGACATGAAGAAAAAACGTTTTGTCTGAATGCTTTCTTGGGTAGTCAGAA ATAAAAGCTGTTGTACGGAAGATCATATGAGGCTGCTATGGGTAGCAGCATCAAGTGTG GCAGTGGAGCAGAGAGAGATTGCATGCCACGGGGAGAGGAAATGTGGAAAATTACACAT ATCACCGTGAG (SEQ ID NO: 5).

[0095] In some embodiments, the recombinant Z chromosome of gender comprising the exogenous reporter gene at site 5a comprises the nucleotide sequence as presented in SEQ ID NO: 5.

[0096] In some embodiments, the recombinant gender Z chromosome comprises the nucleotide sequence as presented in SEQ ID NO: 5, or an analogue thereof, having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identity or homology thereto, or any value and range therebetween. Each possibility represents a distinct embodiment of the invention. In some embodiments, an analogue of the nucleotide sequence as presented in SEQ ID NO: 5, comprises 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 99% to 100% identity or homology with a nucleic acid sequence as presented in SEQ ID NO: 5. Each possibility represents a distinct embodiment of the invention.

[0097] In some embodiments, the recombinant Z chromosome of gender comprises the nucleotide sequence: GGTCTGTGCAAACAGTGTTTCTCATGCAACTTGGCTGCCTTTAAATGGC TTACCAACTCTTTTCTGAAAAACCTAAAAAATGTCTGTGTGCCAAGTAAGATACTTCAA TTCAAAAGGAGGTTTTTCAATTTTTTCTCTCAGTTTATCATTTCTTCTACTTGAAAAAT ATATTTTAATTTTAATGTTTTTTGTTTTATACAAAAATATATGAACTATGTATTACTAT TGTCACCTGTCATTACCAAGAAGTGAATTCCGTTACATAACTTACGGTAAATGGCCCGC CTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACT Petição 870250101549, de 06 / 11 / 2025, pág. 37 / 104 34 / 92 TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCA AGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTA TTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCAT CTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAG CGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGG CGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAA GTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGC GGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGC CGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCC TTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTG GTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTT GGCGCCACCATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCG CATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCC CCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTC GCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCC CGCCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCG TGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGAC GGCTCCTTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGT AATGCAGAAGAAGACTATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACG GCGTGCTGAAGGGCGAGATCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTG GTGGAGTTCAAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTA CGTGGACTCCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGT ACGAGCGCGCCGAGGGCCGCCACCACCTGTTCCTGTAGCGGCCGCGACTCTAGATCATA ATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCC CCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTT ATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCA CTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTGGATCCACATCACCAGGGCTGGAGGTTACATCCTGCTGGCCAAAACTACATTATGGTGGGTTAGCTAA TTTTACAGGAGGGATAAATTGTGAGAATCTGGAAACCATAATCTTGACAAGAAAAAATT Petition 870250101549, dated 06 / 11 / 2025, p. 38 / 104 35 / 92 AACACCCAATTCCTTGGTGAGACTGGGCATTATATGGACATGGGAAATCTCACAGTCAT GATATATGTTAGGAAGGAACTCTCCTTCAAGGGTCCAGGACTGTAAGTATTGGCCTGCC CCAACACTTGG (SEQ ID NO: 6)

[0098] In some embodiments, the recombinant Z chromosome of gender comprising the exogenous reporter gene at site 14a comprises the nucleotide sequence as presented in SEQ ID NO: 6.

[0099] In some embodiments, the recombinant gender Z chromosome comprises the nucleotide sequence as presented in SEQ ID NO: 6, or an analogue thereof, having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identity or homology thereto, or any value and range therebetween. Each possibility represents a distinct embodiment of the invention. In some embodiments, an analogue of the nucleotide sequence as presented in SEQ ID NO: 6, comprises 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 99% to 100% identity or homology with a nucleic acid sequence as presented in SEQ ID NO: 6. Each possibility represents a distinct embodiment of the invention.

[0100] In some embodiments, the recombinant Z chromosome of gender comprises the nucleotide sequence: TGAATGTGGAAAGTGAAGATAACTACTAGTGCAGCAAGCAGACATAGAGATTTTATAAA AGTGGATGGTGAGCATTGTTTGTTAGATACAAACCATTAAGCAGAGATGCCTAGTGACT CAGAGTACAGAGCTTCCTGACTGTCAACACTGACCATGACACTGACCACGTTATGATCC AGGAATAATAGAACCTGTATTTACTCTTAGATATTCTTAAATTGTAATCACAGAAGAGG ACAAACAGTGATAGGAATTCCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACC GCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACTTTCCATTGAC GTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCAT Petição 870250101549, de 06 / 11 / 2025, pág. 39 / 104 36 / 92 ATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTGTGC CCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCG CTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCC TCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGC GGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGG CGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTT ATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGT CGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCC GGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCG GGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATT AATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGCGCCACCA TGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGC TCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGG CACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACA TCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCGCCGACATC CCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTT CGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCTCCTTCA TCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTAATGCAGAAG AAGACTATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAA GGGCGAGATCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCA AGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTGGACTCC AAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCGC CGAGGGCCGCCACCACCTGTTCCTGTAGCGGCCGCGACTCTAGATCATAATCAGCCATA CCACATTTGTAGAGGTTTTACTTGCTTTAAAAACCTCCCACCTCCCCCTGAACCTG AAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTAATGGTTA CAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTA GTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTGGATCCTCTATTGTCTCAT AAGTCCTACCACTCAGGAATAAGGCTTATAAGAAACATGAGAAATGTGTAACTGTAGAG TGAGTTAGACATAGCCAGCAGCTATATACGTACCCATGCTCACAGTCTCTCTCCTTC GCAGGATTAGGAAATAAATTGGGTGAAAAAGGTTATAGATAAGACAGAGATAGAAAGA Petition 870250101549, of 06 / 11 / 2025, p. 40 / 104 37 / 92 TTGCTAATCATTTAACATTTGATTTGGGCAAAGTAATTGAATACACTGTCAAGTAAA G (SEQ ID NO: 39).

[0101] In some embodiments, the recombinant Z chromosome of gender comprising the exogenous reporter gene at site 7b comprises the nucleotide sequence as presented in SEQ ID NO: 39.

[0102] In some embodiments, the recombinant gender Z chromosome comprises the nucleotide sequence as presented in SEQ ID NO: 39, or an analog thereof, having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identity or homology with the same, or any value and range therebetween. Each possibility represents a distinct embodiment of the invention. In some embodiments, an analog of the nucleotide sequence as presented in SEQ ID NO: 39 comprises 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 99% to 100% identity or homology with a nucleic acid sequence as presented in SEQ ID NO: 39. Each possibility represents a distinct embodiment of the invention.

[0103] In some embodiments, the recombinant gender Z chromosome comprises the nucleotide sequence: CAAGAACATTAAAAAGAAACAGGATCTCTCTTGCTTTTGTTAAGAAAAAAAAACAGCAG CAGTACACCAAGAGCAGTTAAATTACTCTATTTAGAGATGTCTGAAACACTTCTAAAGC AAAGCAGGTAATACTTCAGGGACCATCTACAAGCTTGCAGTTTTAGCTCCCTTCTATAA CTTTTCCAATTTGATCTTTCTGTGGAAAAATACAAAATTGTGTCTGTTTCTTGAAGACC GGTAACCAGAAAGTGAATTCCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACC GCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACTTTCCATTGAC GTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCAT Petition 870250101549, dated 06 / 11 / 2025, page 41 / 104 38 / 92 ATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTGTGC CCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCG CTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCC TCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGC GGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGG CGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTT ATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGT CGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCC GGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCG GGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATT AATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGCGCCACCA TGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGC TCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGG CACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACA TCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCGCCGACATC CCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTT CGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCTCCTTCA TCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTAATGCAGAAG AAGACTATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAA GGGCGAGATCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTGGACTCC AAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCGC CGAGGGCCGCCACCACCTGTTCCTGTAGCGGCCGCGACTCTAGATCATAATCAGCCATA CCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTG AAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTA CAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTA GTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTGGATCCTATTTGTCAGAAA AATCTATCTGCATACTCATTTCTTTGAATGAGATTACAATCATGAGATGTCCACTATCT GCATTTTTGATCACTAGTGAAAACTCCTTCTTTCCAAAGCCACTGGTGATTATTGTTTA TTTAGGAGAGCAGGCCTTTAAAAGAAATACTGTGGTCACCTGTGTGACTGAAAAAAGCA Petition 870250101549, dated 06 / 11 / 2025, p. 42 / 104 39 / 92 CATATTCAATTCATGTGAATAACATTAGAAAGTTTCCAGGAACTGCTGATGATTCAAGC A (SEQ ID NO: 40).

[0104] In some embodiments, the recombinant Z chromosome of gender comprising the exogenous reporter gene at site 8a comprises the nucleotide sequence as presented in SEQ ID NO: 40.

[0105] In some embodiments, the recombinant gender Z chromosome comprises the nucleotide sequence as presented in SEQ ID NO: 40, or an analogue thereof, having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identity or homology thereto, or any value and range therebetween. Each possibility represents a distinct embodiment of the invention. In some embodiments, an analogue of the nucleotide sequence as presented in SEQ ID NO: 40, comprises 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 99% to 100% identity or homology with a nucleic acid sequence as presented in SEQ ID NO: 40. Each possibility represents a distinct embodiment of the invention.

[0106] In some embodiments, the recombinant Z chromosome of gender comprises the nucleotide sequence: GAAACATCATTTATAGACTAAGTGTGTATATTATAAATCTGCTGATTGCATAGGTAAAT TTTTTTCAGTATCATCTGCTGTCAAAATTTTGGCTGTGATGAAATCAACTTTCTTCATA GAAACTCATATAATGCTGTGCTTCGGATTTTTGATGAAAATAGTGATGATAATACACCA GTGTTCCAGTTGTGACAGAGCAGTGCTTACAGAGTCAAAAACTTTTTATTTTATTGTGC TATCCTGCCAATGAGAATTCCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACC GCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACTTTCCATTGAC GTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCAT Petição 870250101549, de 06 / 11 / 2025, pág. 43 / 104 40 / 92 ATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTGTGC CCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCG CTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCC TCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGC GGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGG CGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTT ATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGT CGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCC GGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCG GGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATT AATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGCGCCACCA TGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGC TCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGG CACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACA TCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCGCCGACATC CCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTT CGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCTCCTTCA TCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTAATGCAGAAG AAGACTATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAA GGGCGAGATCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCA AGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTGGACTCC AAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCGC CGAGGGCCGCCACCACCTGTTCCTGTAGCGGCCGCGACTCTAGATCATAATCAGCCATA CCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTG AAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTA CAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTA GTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTGGATCCTGCTTTGCTTGCA CATGCAGTAAACTTTTTTAATCTTAATTCATAAACTCTCACACTTTTACATTTCCAATT TTCACATCTTTAATCATCTGGGAGATGATTTCACATCCTCATCAAGTTTGTGGATGACC CAGAACTGAGGGCAGTGGCTAACTCACCAGAGTGCTGTGCTGCCATCTGGAGGAACTTG Petição 870250101549, de 06 / 11 / 2025, pág. 44 / 104 41 / 92 CACAGTTGGAGAAGGGCTGACAGGAACTTGATGGAGTTCTACATGGAGAAGGGAAAGTT C (SEQ ID NO: 41).

[0107] In some embodiments, the recombinant Z chromosome of gender comprising the exogenous reporter gene at site 13a comprises the nucleotide sequence as presented in SEQ ID NO: 41.

[0108] In some embodiments, the recombinant gender Z chromosome comprises the nucleotide sequence as presented in SEQ ID NO: 41, or an analogue thereof, having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identity or homology thereto, or any value and range therebetween. Each possibility represents a distinct embodiment of the invention. In some embodiments, an analogue of the nucleotide sequence as presented in SEQ ID NO: 41, comprises 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 99% to 100% identity or homology with a nucleic acid sequence as presented in SEQ ID NO: 41. Each possibility represents a distinct embodiment of the invention.

[0109] According to another aspect, a cell obtained or derived from the transgenic female chicken disclosed in the present invention is provided. In some embodiments, the cell comprises a recombinant Z chromosome, as disclosed in the present invention. In some embodiments, the exogenous reporter gene, encoding RFP, is integrated into the cell's Z chromosome. In some embodiments, the cell comprises a primordial germ cell (PGC). In some embodiments, the cell is a PGC. In some embodiments, a PGC comprising a Petition 870250101549, dated 06 / 11 / 2025, page 45 / 104 42 / 92 nucleic acid sequence of an exogenous reporter gene encoding RFP, integrated into the Z chromosome of the same genus.

[0110] The term “germ cell” refers to an embryonic cell with the potential to develop into a gamete. The term “primordial germ cell (PGC)”, as used in the present invention, refers to a germline stem cell that serves as a progenitor of a gamete and gives rise to a pluripotent embryonic stem cell.

[0111] The use of PGCs to produce transgenic chickens is well known in the art. Early-stage PGCs are readily accessible and can be manipulated in vitro for practical applications, including genetic material restoration and genome editing. The ability of chicken PGCs to be maintained undifferentiated in vitro without losing their properties, as well as their high migratory capacity through the bloodstream of the chicken embryo, highlight PGCs as a leading source for transgenesis. In some embodiments, PGCs are used to produce a chimeric and / or transgenic chicken by injecting genetically manipulated PGCs into the blood vessels of a recipient egg. However, it is noted that other cells are known in the art for the generation of a transgenic chicken. Non-limiting examples of such cells include embryonic stem cells (ESCs) and spermatogonial stem cells (SSCs).

[0112] According to another aspect, a method is provided for producing a chicken comprising a recombinant Z chromosome of gender, the method comprising: (a) obtaining at least one transgenic PGC comprising a chromosome Petition 870250101549, dated 06 / 11 / 2025, page 46 / 104 43 / 92 (a) a recombinant Z chromosome comprising a nucleic acid sequence of an exogenous reporter gene encoding an RFP at least one location as listed in Table 1 on a chicken's gender Z chromosome; and (b) transplanting the transgenic PGC into a receptive chicken embryo, thereby producing a chicken comprising the gender-recombinant Z chromosome.

[0113] In some modalities, the transplant involves injecting the transgenic PGCs into at least one blood vessel of a recipient egg.

[0114] In some embodiments, the recombinant Z chromosome comprises a nucleic acid sequence of an exogenous reporter gene encoding an RFP at at least one location as listed in Table 3. In some embodiments, the at least one location is selected from: site 4a, site 5a, site 13a, site 14a, or any combination thereof.

[0115] In some embodiments, the method disclosed in the present invention is for producing a chimeric or transgenic chicken. Note that steps (a) and (b), as disclosed in the present invention, refer only to the steps necessary to obtain a chicken comprising the recombinant Z-gender chromosome, such as, but not limited to, a chimeric chicken. Other steps applicable to the generation of a transgenic chicken and protocols thereof (for example, protocols for generating germline chimeras and screening techniques for the presence of the recombinant Z-chromosome) are well known in the art. Some of these protocols are disclosed in document WO2017094015A1, which Petition 870250101549, dated 06 / 11 / 2025, page 47 / 104 44 / 92 is incorporated into the present invention by reference in its entirety.

[0116] In some embodiments, the method further comprises a step prior to step (a), comprising isolating at least one PGC from the blood of a chick embryo. In one embodiment, a chick embryo is a two-day-old embryo. In some embodiments, a chick embryo is a naive chick embryo. In some embodiments, a chick embryo is a two-day-old naive chick embryo.

[0117] As used in the present invention, the term “naive” refers to an embryo having a genome lacking a recombinant Z chromosome of gender as disclosed in the present invention. In some embodiments, naive refers to an embryo having a genome lacking a nucleic acid sequence of an exogenous reporter gene encoding RFP as disclosed in the present invention.

[0118] In some embodiments, the method further comprises a step prior to step (a), comprising integrating the nucleic acid sequence of an exogenous reporter gene encoding an RFP at least one location as listed in Table 1, on the Z chromosome of the PGC genus, thereby obtaining a transgenic PGC. In some embodiments, the integration of the nucleic acid sequence of an exogenous reporter gene encoding an RFP as disclosed in the present invention is performed using a CRISPR / Cas9 system. In some embodiments, the integration of the nucleic acid sequence of an exogenous reporter gene encoding an RFP is performed using a CRISPR / Cas9 system comprising gRNA, Petition 870250101549, dated 06 / 11 / 2025, page 48 / 104 45 / 92 comprising a nucleic acid sequence as presented in any of the SEQ ID Nos: 1 - 3, and 18 20.

[0119] In some embodiments, transplanting transgenic PGC into a receptive chicken embryo in step (b) involves injecting PGC into the circulatory system of the receptive chicken embryo.

[0120] In some embodiments, the method for producing a chicken comprising a recombinant gender Z chromosome further comprises one or more steps selected from: (c) incubating the transplanted embryo with PGC at 36 to 38 °C until hatching; (d) raising a chick that develops from the transplanted embryo of step (c) to sexual maturity; (e) crossing the sexually mature chicken that develops from the chick of step (d) with a matching counterpart; (e) isolating at least one offspring from a progeny obtained by crossing step (e) comprising the recombinant gender Z chromosome, or any combination thereof. In some embodiments, the crossing comprises at least one cross. In some embodiments, the crossing comprises a plurality of crossing steps to generate a transgenic chicken disclosed in the present invention.

[0121] In some embodiments, the transgenic PGC comprising a recombinant Z chromosome is a female PGC, for example, obtained from a female chick embryo. In some embodiments, the transgenic female PGC is transplanted into a receptive female chicken embryo, thus producing a female chicken comprising the recombinant Z gender chromosome. In some embodiments, a chick that is Petition 870250101549, dated 06 / 11 / 2025, page 49 / 104 46 / 92 develops from the transplanted embryo and is a chimeric female chick comprising the recombinant Z gender chromosome. In some embodiments, the chimeric female chick is additionally crossed with a corresponding male. In some embodiments, the corresponding male is a wild male.

[0122] In some embodiments, the transgenic PGC comprising a recombinant Z chromosome is a male PGC, for example, obtained from a male chick embryo. In some embodiments, the transgenic male PGC is transplanted into a receptive male chicken embryo, thus producing a male chicken comprising the recombinant Z gender chromosome. In some embodiments, a chick that develops from the transplanted embryo is a chimeric male chick comprising the recombinant Z gender chromosome. In some embodiments, the chimeric male chicken is further crossed with a matching female. In some embodiments, the matching female is a wild-type female.

[0123] In some embodiments, a method is provided for producing a male chicken comprising at least one recombinant Z gender chromosome as disclosed in the present invention, the method comprising obtaining at least one male offspring from the chimeric male or female chicken disclosed in the present invention. In some embodiments, the male chick comprises two recombinant Z gender chromosomes. In some embodiments, the method further comprises a step comprising isolating or selecting a male chick comprising two recombinant Z gender chromosomes, as disclosed in the present invention. Petition 870250101549, dated 06 / 11 / 2025, page 50 / 104 47 / 92 invention. In some embodiments, the method further comprises obtaining at least one female offspring from a male chicken comprising two recombinant Z-gender chromosomes, as disclosed in the present invention, thereby obtaining a transgenic female chicken comprising one recombinant Z-gender chromosome, as disclosed in the present invention. Sex Determination Method

[0124] According to another aspect, a method is provided for determining the sex of a chicken embryo in an unhatched egg comprising the embryo within a structurally intact shell, the method comprising: (a) obtaining at least one unhatched egg comprising an embryo within a structurally intact shell, from a transgenic female chicken as disclosed in the present invention; and (b) determine whether a red fluorescent signal is detected in the embryo residing in the unhatched egg.

[0125] In some embodiments, detection of the red fluorescent signal indicates RFP expression in the embryo within the structurally intact shell of the unhatched egg and thus indicates the presence of the recombinant Z chromosome in the embryo, thus determining that the chicken embryo in the unhatched egg is a male embryo.

[0126] In some embodiments, the absence of detection of the red fluorescent signal in the resident embryo in the unhatched egg indicates that RFP is not expressed in the embryo and thus indicates the absence of the recombinant Z chromosome in the embryo, thus determining that the chicken embryo in the unhatched egg is a female embryo. Petition 870250101549, dated 06 / 11 / 2025, page 51 / 104 48 / 92

[0127] In some embodiments, a transgenic female chicken comprises a recombinant Z chromosome comprising the nucleotide sequence presented in any of the SEQ ID Nos: 4 - 6, and 39 - 41, or an analogue thereof, as disclosed in the present invention.

[0128] In some embodiments, the method additionally comprises a step prior to step (b), comprising isolating at least one unhatched egg determined to contain a female embryo, for example, a red fluorescent signal is not detected, thus isolating at least one female embryo.

[0129] As used in the present invention, the term “structurally intact shell” refers to the shell of an egg that is not: structurally damaged, cracked, broken, hatched, perforated, punctured, thinned, or any combination thereof. It should be understood that the gender determination method disclosed in the present invention may be applicable to unhatched eggs of any stage of embryonic development of a chicken embryo.

[0130] The term “embryonic development stage of a chicken embryo”, as used in the present invention, refers to the day 1 stage, in which the germinal disc is at the blastodermic stage and the segmentation cavity takes the form of a dark ring; the day 2 stage, in which the first furrow appears in the center of the blastoderm and the vitelline membrane appears; the day 3 stage, in which blood circulation begins, the head and trunk can be discerned, as well as the brain and cardiac structures that begin to beat; the day 4 stage, in which the amniotic cavity is developing to enclose the Petition 870250101549, dated 06 / 11 / 2025, page 52 / 104 49 / 92 embryo and the allantoic vesicle appear; day 5 stage, in which the embryo assumes a C shape and the limbs are extending; day 6 stage, in which the fingers of the upper and lower limbs become distinct; day 7 stage, in which the neck clearly separates the head from the body, the beak is formed and the brain progressively enters the cephalic region; day 8 stage, in which the pigmentation of the eyes is easily visible, the wings and legs are differentiated and the external auditory canal is opening; day 9 stage, in which the claws appear and the first feather follicles are sprouting; day 10 stage, in which the nostrils are present, the eyelids grow and the egg tooth appears; day 11 stage, in which the palpebral opening has an elliptical shape and the embryo has the appearance of a chick; the 12-day stage in which the feather follicles surround the external auditory meatus and cover the upper eyelid, while the lower eyelid covers most of the cornea; The 13th stage, in which the allantois becomes the chorioallantoic membrane while the claws and leg scales become apparent; the stages from days 14 to 16, in which the entire body grows rapidly, the calf's shrinkage accelerates, and the egg white progressively disappears; the 17th stage, in which the renal system produces urates, the beak points towards the air cell, and the egg white is completely reabsorbed; the 18th stage, when the calf is internalized and the amount of amniotic fluid is reduced; the 19th stage, in which the calf's reabsorption accelerates and the beak is ready to pierce the inner shell membrane; the 20th stage, when the calf is completely reabsorbed, the navel is closed, and the chick pierces the Petition 870250101549, dated 06 / 11 / 2025, pp. 53 / 104 50 / 92 inner membrane of the shell, breathes in the air cell and is ready to hatch; the 21st day stage, in which the chick pierces the shell in a circular motion using its egg tooth, detaches from the shell in 12 to 18 hours and lets its down dry.

[0131] In some embodiments, the method comprises determining the sex of a chicken embryo in ovo, within an intact egg, while it resides in an egg with an intact or whole shell, or any combination thereof, at each stage of the embryonic development process. In some embodiments, the method comprises determining the sex of a chicken embryo from day 1 to day 21, from day 1 to day 20, from day 1 to day 19, from day 1 to day 18, from day 1 to day 17, from day 1 to day 16, from day 1 to day 15, from day 1 to day 14, from day 1 to day 13, from day 1 to day 12, from day 1 to day 10, from day 1 to day 9, from day 1 to day 8, from day 1 to day 7, from day 1 to day 6, and from day 1 to day 5. Each possibility represents a separate embodiment of the present invention.

[0132] In some embodiments, the method further comprises a step comprising subjecting the unhatched egg comprising the embryo within a structurally intact shell to a light source.

[0133] In some embodiments, the light source is applicable or configured to detect RFP. In some embodiments, the light source comprises a wavelength between about 400 nm and about 650 nm. In some embodiments, the light source comprises a wavelength between 500 nm and about 650 nm. In some embodiments, the light source comprises a wavelength ranging Petition 870250101549, dated 06 / 11 / 2025, p. 54 / 104 51 / 92 between approximately 515 and approximately 555. In some modalities, the light source comprises a wavelength range of 500 - 600, 510 - 600, 520 - 600, 520 - 600, 530 - 600, 500 - 590, 510 - 590, 520 - 590, 530 - 590, 500 - 580, 510 - 580, 520 - 580, 530 - 580, 500 - 570, 510 - 570, 520 - 570, 530 - 570, 500 - 560, 510 - 560, 530 - 560, 500 - 550, 510 - 550, 520 - 550, 530 - 550, 500 - 540, 510 - 540, 510 - 540, 520 - 540, or 530 - 540nm. Each possibility represents a distinct embodiment of the invention. In some additional embodiments, the wavelength may be approximately 532 nm.

[0134] In some specific and non-limiting modalities, the light source may be provided by a laser.

[0135] As used in the present invention, the term laser refers to electromagnetic radiation of any frequency that is amplified by stimulated emission of radiation. A laser also refers to a device that stimulates atoms or molecules to emit light at specific wavelengths and amplifies that light, typically producing a very narrow beam of radiation. In some embodiments, the light source is a green laser.

[0136] In some embodiments, the unhatched egg is exposed to the light source. In some embodiments, the egg is placed in a position that allows the embryo to be exposed to the light source at any stage. In some embodiments, a region containing the upper face of the egg yolk at stage X of the egg is excited with the light source.

[0137] In some additional embodiments, the step of subjecting the unhatched egg to a light source is provided by a system, apparatus or device that may comprise Petition 870250101549, dated 06 / 11 / 2025, p. 55 / 104 52 / 92 a laser source, an egg holder, a lens, a filter, a detector holder and a detector.

[0138] As used in the present invention, the term detector refers to any type of device that detects and / or measures light. In some embodiments, it should be noted that the detectable signal, specifically the fluorescent signal, can be detected using suitable fluorescent media. In some embodiments, the detectable signal formed by the exogenous RFP reporter gene can be detected by light-sensitive instruments such as modified optical microscopes or Charge-Coupled Devices (CCDs), a highly sensitive photon detector.

[0139] It is noted that, in some embodiments, the method additionally comprises a step, a system or device for its execution, necessary for the detection of RFP in the unhatched egg examined. Examples of such steps, systems and / or devices are further described in detail in document WO2017094015A1, which is incorporated into the present invention by reference in its entirety. Kits

[0140] According to another aspect, a kit is provided comprising at least one first nucleic acid molecule encoding a guide RNA (gRNA), comprising the nucleotide sequence as presented in any of the SEQ ID Nos: 1 - 3, and 18 - 20.

[0141] In some embodiments, the kit additionally comprises at least one second nucleic acid molecule that encodes: Cas9 protein, an RFP, or both.

[0142] In some embodiments, the kit comprises a first nucleic acid molecule that encodes a gRNA. Petition 870250101549, dated 06 / 11 / 2025, page 56 / 104 53 / 92 as disclosed in the present invention, a second nucleic acid molecule encoding a Cas9 protein, and a third nucleic acid molecule encoding an RFP.

[0143] In some embodiments, the nucleic acid molecule as disclosed in the present invention is integrated into or resides in at least one expression vector or plasmid.

[0144] In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule are integrated into or reside in the same expression vector or plasmid. In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule are integrated into or reside in a first expression vector or plasmid and the third nucleic acid molecule is integrated into or resides in a second expression vector or plasmid.

[0145] In some, the kit comprises: (i) at least one first nucleic acid molecule encoding a gRNA, comprising the nucleotide sequence as presented in any of the SEQ ID Nos: 1 - 3, and 18 - 20, (ii) a second nucleic acid molecule comprising a sequence encoding Cas9 protein; and (iii) a third nucleic acid molecule comprising a sequence encoding RFP, as disclosed in this invention.

[0146] In some embodiments, the second nucleic acid molecule encoding Cas9 protein comprises the nucleotide sequence: ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAA GTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACG Petition 870250101549, dated 06 / 11 / 2025, p. 57 / 104 54 / 92 AGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATC AAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCG GCTGAAGGAACCGCCAGAAGAAGATACACCAGACGGAAACCGGATCTGCTATCTGC AAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAA GAGTCCTTCCTGGTGGAAGAGGATAAGACGAGCGGCACCCATCTTCGGCAACAT CGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAAC TGGTGGAACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATG ATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGT GGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCA TCAACGCCAGCGGCTGGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGA CGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAA CCTGATTGCCCTGAGCCTGGGCCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCG AGGATGCCAAACTGCCAGTGAGCAAGGACACCTACGACAGCTGGACAACCTGCTGCGCGCCAGAAGCTTCCGACGC CATCCTGCTGAGCGACATCCTGAGTCCGAGAGTGGAACACCAGAGATCACCAAGGCCCCCTGAGC CCTCTATGATCAAGAGATACGACGAGCCACCCAGGACCTGACCTGCTGAAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGG CTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGC CCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGAC CTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGG AGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACC GGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCC AGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTG GAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGA CCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTAC GAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAG AAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGA CCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGC TTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATA CCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGG Petição 870250101549, de 06 / 11 / 2025, pág. 58 / 104 55 / 92 ACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAG GAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCG GCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACA AGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAAC TTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCA GGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCG CCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATG GGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCA GAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGC TGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAG CTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACAT CAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACG ACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAAC GTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGC CAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGA GCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAA GCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGA AGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCC TACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGA GTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGC AGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTC AAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAA CGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAG TGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTC AGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGA CTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGG TGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTG GGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGC CAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGT Petição 870250101549, de 06 / 11 / 2025, pág. 59 / 104 56 / 92 TCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGA AACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGA GAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACA AGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTG GCCGAGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCAT CAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTG CCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAG GTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGA CCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAA AAAAGAAAAAGTAA (SEQ ID NO: 9), or an analogue thereof, having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identity or homology thereto, or any value and range thereof. Each possibility represents a distinct embodiment of the invention.In some embodiments, an analog of the second nucleic acid molecule encoding Cas9 comprises 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 99 to 100% identity or homology with a nucleic acid sequence as presented in SEQ ID NO: 9. Each possibility represents a distinct embodiment of the invention.

[0147] In some embodiments, the third nucleic acid molecule that encodes RFP comprises a nucleic acid sequence as presented in SEQ ID NO: 8, or an analog thereof as disclosed in this invention.

[0148] In some embodiments, the third nucleic acid molecule comprises a promoter. In some embodiments, the promoter comprises a chicken β-actin (CBA) promoter. In some embodiments, the promoter comprises a CBh promoter. In some embodiments, the promoter comprises the nucleotide sequence: Petition 870250101549, dated 06 / 11 / 2025, pp. 60 / 104 57 / 92 GAATTCCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCC GCCCATTGACGTCAATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAG TATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCC CCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTGTGCCCAGTACATGACCT TATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTC GAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAAT TTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGG GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGT GCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCG GCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCC TTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACC GCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCT GAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACC TGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGCGCCACC (SEQ ID NO: 10).

[0149] In some embodiments, the third nucleic acid molecule comprises a nucleotide sequence that encodes a polyadenylation (polyA) signal. In some embodiments, a nucleotide sequence that encodes a polyA signal comprises the nucleotide sequence: CGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTT AAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTG TTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTC ACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGT ATCTTAAGGCGTGGATCC (SEQ ID NO: 11).

[0150] In some embodiments, the third nucleic acid molecule comprises a nucleotide sequence that allows the integration of the exogenous reporter gene at least one location on a gender Z chromosome. In some embodiments, SEQ ID NO: 8, or an analogue thereof, is Petition 870250101549, dated 06 / 11 / 2025, pp. 61 / 104 58 / 92 flanked at 5' of the same, 3' of the same, and both by a homologous arm. In some embodiments, the integration of the exogenous reporter gene at least at one location on a Z-gender chromosome occurs by homology-directed repair (HDR) of an arm homologous to a sequence within the genomic site disclosed in the present invention.

[0151] The term “homology-directed repair (HDR),” as used in the present invention, encompasses a process in which a DNA double-strand break (DSB) is repaired by homologous recombination using a DNA template. The term “homologous arm,” as used in the present invention, refers to an HDR template introduced into a specific vector or plasmid, designed to align a functional gene to a specific site in the genome. In some embodiments, where CRISPR is used as a PEN, the arm sequences (e.g., 5' homology arm; left, upstream, and 3' homology arm; right, downstream) comprise between about 10 and 5,000 bp, between about 50 and 1,000 bp, or between about 100 and 500 bp.

[0152] In some embodiments, the third nucleic acid molecule comprises a 5' homology arm. In some embodiments, the 5' homology arm comprises the nucleotide sequence: GGCCTATACAGACAAATCTTTACTAAATGGAGAATCCTGACATTTTGTCCATTTCTCTC TATACCACCAACTCTCATCTTGGTTACATTGCCTGTCAGTTGAACAGGCAAGATCTCCA TTCCCAAAACACCACACTTATCTTGAATTGAGCCATCAAGAGGTATTTCTCTGATACAC CATTATCTAAAACCCAGTGTTATTTTTCAGTGAGGGTAGATTCCCTTTACTGCTTTTTC TCCTCATTTCCA (SEQ ID NO: 12).

[0153] In some embodiments, the third nucleic acid molecule comprises a 3' homology arm. In Petition 870250101549, dated 06 / 11 / 2025, pp. 62 / 104 59 / 92 In some forms, the 3' homology arm comprises the nucleotide sequence: ATAACGGCATTCAGTACTTGTCATCACTAATTAGTGTATTCAATCTACCCTCTATTTCT AGGACAACATGTTTTAATAATTCACCATAAATTAGTTCTATGAGCATTTTGATCTAGTG GTTGTTGTAGATCACAGGATATGAATAGATTGTGTGTATGTGTAAACCAGGAGTGTCTT GACAAAAGGACACAGTGCTGGTAAGACCAGCAGAAGAGGGTCCCCTGTGGCAAAAATGT TTTACTGACCTTTG (SEQ ID NO: 13).

[0154] In some embodiments, a third nucleic acid molecule comprising a nucleotide sequence as presented in: SEQ ID NO: 12, SEQ ID NO: 13, and both, is used to integrate the exogenous reporter gene into site 4a.

[0155] In some embodiments, the 5' homology arm comprises the nucleotide sequence: AATACACAGCACTGTATCTTTTATGGCAATCTATTTTCGATTGTCTCATGTGCAGAACA GTTACCAGGATTGCAACAACGGATTGAATTTATCCGAAAAGAAGTTTTGTGTCCTGCTT TGTGATAGCTGAGAAAGAAAGGCAGTGATGCTTAAAAAGCAGTCAGTGACCTAATCACC TACTGTCAGGTGTACTATGAATACATACAGTAGAGCCAGTAACACAGTTTGACAGCATT TTCATTAGATGTTT (SEQ ID NO: 14).

[0156] In some embodiments, the 3' homology arm comprises the nucleotide sequence: CAGAACAAAATAGTATTTTTGTTCACAACTGGGAGTGAAATCTGATTTCAAACCACTAA AAAGAATAGTGGAGACATGAAGAAAAAACGTTTTGTCTGAATGCTTTCTTGGGTAGTCA GAAATAAAAGCTGTTGTACGGAAGATCATATGAGGCTGCTATGGGTAGCAGCATCAAGT GTGGCAGTGGAGCAGAGAGAGATTGCATGCCACGGGGAGAGGAAATGTGGAAAATTACA CATATCACCGTGAG (SEQ ID NO: 15).

[0157] In some embodiments, a third nucleic acid molecule comprising a nucleotide sequence as presented in: SEQ ID NO: 14, SEQ ID NO: Petition 870250101549, dated 06 / 11 / 2025, pp. 63 / 104 60 / 92 15, and both, is used to integrate the exogenous reporter gene into site 5a.

[0158] In some modalities, the homology arm 5' comprises the nucleotide sequence: GGTCTGTGCAAACAGTGTTTCTCATGCAACTTGGCTGCCTTTAAATGGCTTACCAACTC TTTTCTGAAAAACCTAAAAAATGTCTGTGTGCCAAGTAAGATACTTCAATTCAAAAGGA GGTTTTTCAATTTTTTCTCTCAGTTTATCATTTCTTCTACTTGAAAAATATATTTTAAT TTTAATGTTTTTTGTTTTATACAAAAATATATGAACTATGTATTACTATTGTCACCTGT CATTACCAAGAAGT (SEQ ID NO: 16).

[0159] In some modalities, the homology arm 3' comprises the nucleotide sequence: ACATCACCAGGGCTGGAGGTTACATCCTGCTGGCCAAAACTACATTATGGTGGGTTAGC TAATTTTACAGGAGGGATAAATTGTGAGAATCTGGAAACCATAATCTTGACAAGAAAAA ATTAACACCCCCAATTCCTTGGTGAGACTGGGCATTATATGGACATGGGAAATCTCCACAGT CATGATATATGTTAGGAAGGAACTCTCCTTCAAGGGTCCAGGACTGTAAGTATTGGCCT GCCCCAACACTTGG (SEQ ID NO: 17).

[0160] In some embodiments, a third nucleic acid molecule comprising a nucleotide sequence as presented in: SEQ ID NO: 16, SEQ ID NO: 17, and both, is used to integrate the exogenous reporter gene into site 14a.

[0161] In some embodiments, the 5' homology arm comprises the nucleotide sequence: TGAATGTGGAAAGTGAAGATAACTACTAGTGCAGCAAGCAGACATAGAGATTTTATAAA AGTGGATGGTGAGCATTGTTTGTTAGATACAAACCATTAAGCAGAGATGCCTAGTGACT CAGAGTACAGAGCTTCCTGACTGTCAACACTGACCATGACACTGACCACGTTATGATCC AGGAATAATAGAACCTGTATTTACTCTTAGATATTCTTAAATTGTAATCACAGAAGAGG ACAAACAGTGATAG (SEQ ID NO: 21).

[0162] In some embodiments, the 3' homology arm comprises the nucleotide sequence: Petition 870250101549, dated 06 / 11 / 2025, pp. 64 / 104 61 / 92 TCTATTGTCTCATAAGTCCTACCACTCAGGAATAAGGCTTATAAGAAACATGAGAAATG TGTAACTGTAGAGTGAGTTAGACATAGCCAGCAGCTATATACGTACCCATATGCTCACA GTCTCTCTCCTTCGCAGGATTAGGAAATAAAATTGGGTGAAAAAGGTTATAGATAAGAC AGAGATAGAAAGATTGCTAATCATTTAACATATTTGATTTGGGCAAAGTAATTGAATAC ACTGTCAAGTAAAG (SEQ ID NO: 22).

[0163] In some embodiments, a third nucleic acid molecule comprising a nucleotide sequence as presented in: SEQ ID NO: 21, SEQ ID NO: 22, and both, is used to integrate the exogenous reporter gene into site 7b.

[0164] In some embodiments, the 5' homology arm comprises the nucleotide sequence: CAAGAACATTAAAAAGAAACAGGATCTCTCTTGCTTTTGTTAAGAAAAAAAAACAGCAG CAGTACACCAAGAGCAGTTAAATTACTCTATTTAGAGATGTCTGAAACACTTCTAAAGC AAAGCAGGTAATACTTCAGGGACCATCTACAAGCTTGCAGTTTTAGCTCCCTTCTATAA CTTTTCCAATTTGATCTTTCTGTGGAAAAATACAAAATTGTGTCTGTTTCTTGAAGACC GGTAACCAGAAAGT (SEQ ID NO: 23).

[0165] In some embodiments, the 3' homology arm comprises the nucleotide sequence: TATTTGTCAGAAAAATCTATCTGCATACTCATTTCTTTGAATGAGATTACAATCATGAG ATGTCCACTATCTGCATTTTTGATCACTAGTGAAAACTCCTTCTTTCCAAAGCCACTGG TGATTATTGTTTATTTAGGAGAGCAGGCCTTTAAAAGAAATACTGTGGTCACCTGTGTG ACTGAAAAAAGCACATATTCAATTCATGTGAATAACATTAGAAAGTTTCCAGGAACTGC TGATGATTCAAGCA (SEQ ID NO: 24).

[0166] In some embodiments, a third nucleic acid molecule comprising a nucleotide sequence as presented in: SEQ ID NO: 23, SEQ ID NO: 24, and both, is used to integrate the exogenous reporter gene into site 8a. Petition 870250101549, dated 06 / 11 / 2025, pp. 65 / 104 62 / 92

[0167] In some modalities, the homology arm 5' comprises the nucleotide sequence: GAAACATCATTTATAGACTAAGTGTGTATATTATAAATCTGCTGATTGCATAGGTAAAT TTTTTTCAGTATCATCTGCTGTCAAAATTTTGGCTGTGATGAAATCAACTTTCTTCATA GAAACTCATATAATGCTGTGCTTCGGATTTTTGATGAAAATAGTGATGATAATACACCA GTGTTCCAGTTGTGACAGAGCAGTGCTTACAGAGTCAAAAACTTTTTATTTTATTGTGC TATCCTGCCAATGA (SEQ ID NO: 25).

[0168] In some modalities, the homology arm 3' comprises the nucleotide sequence: TGCTTTGCTTGCACATGCAGTAAACTTTTTTAATCTTAATTCATAAACTCTCACACTTT TACATTTCCAATTTTCACATCTTTAATCATCTGGGAGATGATTTCACATCCTCATCAAG TTTGTGGATGACCCAGAACTGAGGGCAGTGGCTAACTCACCAGAGTGCTGTGCTGCCAT CTGGAGGAACTTGCACAGTTGGAGAAGGGCTGACAGGAACTTGATGGAGTTCTACATGG AGAAGGGAAAGTTC (SEQ ID NO: 26).

[0169] In some embodiments, a third nucleic acid molecule comprising a nucleotide sequence as presented in: SEQ ID NO: 25, SEQ ID NO: 26, and both, is used to integrate the exogenous reporter gene into site 13a.

[0170] In some embodiments, at least one first nucleic acid molecule and any one of the following: the second nucleic acid molecule and the third nucleic acid molecule, are operably linked. In some embodiments, at least one first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule disclosed in the present invention are operably linked. In some embodiments, at least one first nucleic acid molecule and the second nucleic acid molecule are operably linked. Petition 870250101549, dated 06 / 11 / 2025, pp. 66 / 104 63 / 92

[0171] The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0172] In some embodiments, the kit additionally comprises instructions for integrating the at least second nucleic acid molecule encoding RFP into at least one gender Z chromosome location of a female chicken, wherein the at least one location is listed in Table 1. In some embodiments, the at least one location is listed in Table 3. In some embodiments, the at least one location is selected from: site 4a, site 5a, site 13a, site 14a, or any combination thereof.

[0173] The term “encoding” is intended to mean that the nucleic acid in question can be transcribed and translated into the desired polypeptide or protein in question in an appropriate expression system, for example, when the nucleic acid in question is linked to appropriate control sequences, such as promoter and enhancer elements, in a suitable vector (e.g., an expression vector) and when the vector is introduced into an appropriate system or cell. The term “nucleic acid” is intended to mean natural and / or synthetic linear, circular and sequential arrangements of nucleotides and nucleosides, for example, cDNA, genomic DNA (gDNA), mRNA and RNA, oligonucleotides, oligonucleosides and derivatives thereof. Petition 870250101549, dated 06 / 11 / 2025, pp. 67 / 104 64 / 92

[0174] It should be noted that, in some embodiments, at least one of the first and second nucleic acid sequences provided and used by the methods and kits of the invention can be constructed and comprised in a vector. Vectors, as used in the present invention, encompass vectors such as plasmids, phagemids, viruses, integrable DNA fragments, and other vehicles, which allow the integration of DNA fragments into the host genome or, alternatively, allow the expression of non-integrated genetic elements. Vectors are typically self-replicating DNA or RNA constructs containing the desired nucleic acid sequences and operationally linked genetic control elements that are recognized in a suitable host cell and effect the translation of the desired spacers. Generally, the genetic control elements may include a prokaryotic promoter system or a eukaryotic promoter expression control system.Such a system typically includes a transcriptional promoter and transcription enhancers to raise the level of RNA expression. Vectors generally contain an origin of replication that allows the vector to replicate independently of the host cell. In some alternative embodiments, the expression vectors used by the invention may comprise elements necessary for the integration of the desired exogenous reporter gene into the chicken genus-specific Z chromosome.

[0175] Consequently, the term control and regulation elements includes promoters, terminators, and other expression control elements. Such regulatory elements are described in Goeddel; [Goeddel., et al., Gene Petition 870250101549, dated 06 / 11 / 2025, pp. 68 / 104 65 / 92 [Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990)]. For example, any one of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively bound to it can be used in these vectors to express DNA sequences encoding any desired protein using the method of this invention.

[0176] The term “promoter” as used in the present invention refers to a group of transcriptional control modules that are clustered around the initiation site of an RNA polymerase, namely RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7 to 20 bp of DNA and containing one or more recognition sites for transcriptional activator or repressor proteins. The promoter may extend upstream or downstream of the transcription start site and may be of any size, ranging from a few base pairs to several kilobases. In some embodiments, the promoter is a chicken cell promoter. In some embodiments, the promoter is present on the Z chromosome of a chicken cell. In some embodiments, the promoter is an inducible promoter.

[0177] As used in the present invention, the term “inducible promoter” encompasses a promoter that is activated only by a specific stimulus. Once activated, the inducible promoter binds to RNA polymerase and transcription factors, enabling the transcription process.

[0178] A vector may additionally include appropriate restriction sites, antibiotic resistance, or other markers for selecting cells containing the vector. Petition 870250101549, dated 06 / 11 / 2025, pages 69 / 104 66 / 92 Plasmids are the most commonly used vector form, but other vector forms that perform an equivalent function and that are, or have become, known in the art are suitable for use in the present invention. See, for example, Pouwels et al., Cloning Vectors: a Laboratory Manual (1985 and supplements), Elsevier, NY; and Rodriguez, et al. (eds.) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston, Mass (1988), which are incorporated into the present invention by reference.

[0179] In some embodiments, the vector is introduced into the cell by standard methods, including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 L1985)), heat shock, viral vector infection, high-velocity ballistic penetration by small particles with nucleic acid, either within the array of small spheres or particles, or on the surface (Klein et al., Nature 327. 7073 L1987)), such as the biolistic use of coated particles and needle-shaped particles, Agrobacterium Ti plasmids and / or the like.

[0180] In some embodiments, the nucleic acid molecule is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells, known to catalyze the transcription of DNA to synthesize mRNA precursors and most snRNA and microRNA.

[0181] It should be noted that, in addition to containing the elements necessary for the transcription and translation of the inserted coding sequence (which encodes the polypeptides disclosed in the present invention), the construction Petition 870250101549, dated 06 / 11 / 2025, pp. 70 / 104 67 / 92 expression of the present invention may also include sequences designed to optimize the stability, production, purification, yield, or activity of the expressed polypeptide.

[0182] Any concentration ranges, percentage ranges or ratio ranges mentioned in the present invention shall be understood as including concentrations, percentages or ratios of any whole number within that range and fractions thereof, such as one tenth and one hundredth of a whole number, unless otherwise indicated.

[0183] Any numerical range mentioned in the present invention relating to any physical feature, such as polynucleotides and polypeptides, size, weight or length, should be understood as including any integer within the mentioned range, unless otherwise indicated.

[0184] In the discussion, unless otherwise indicated, adjectives such as substantially and about which modify a characteristic condition or relationship of a feature or features of an embodiment of the invention are understood to mean that the condition or characteristic is defined within acceptable tolerances for the operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word or in the descriptive report and claims is considered the inclusive or rather the exclusive or, and indicates at least one of, or any combination of, items that unites.

[0185] It should be understood that the terms "a" and "an," as used above and elsewhere in this document, refer to one or more of the enumerated components. It will be Petition 870250101549, dated 06 / 11 / 2025, pp. 71 / 104 68 / 92 It is clear to anyone with common knowledge of the technique that the use of the singular includes the plural, unless specifically indicated otherwise. Therefore, the terms "a," "an," and "at least one" are used interchangeably in this order.

[0186] The descriptions of the various embodiments of the present invention have been presented for illustrative purposes, but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be evident to those with common knowledge of the art, without departing from the scope and spirit of the embodiments described. The terminology used in the present invention has been chosen to better explain the principles of the embodiments, the practical application or the technical improvement in relation to technologies found on the market, or to allow others with common knowledge of the art to understand the embodiments disclosed in the present invention.

[0187] For the purposes of better understanding of the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values ​​used in the descriptive report and claims, shall be understood as being modified in all cases by the term approximately. Consequently, unless otherwise indicated, the numerical parameters set forth in the specification and claims appended below are approximations that may vary depending on the desired properties. At a minimum, each numerical parameter shall be interpreted in light of the number of significant digits. Petition 870250101549, dated 06 / 11 / 2025, pp. 72 / 104 69 / 92 reported and by applying common rounding techniques.

[0188] In the description and claims of the present application, each of the verbs, comprehend, include and have, and conjugations thereof, are used to indicate that the object or objects of the verb are not necessarily a complete list of components, elements, or parts of the subject or subjects of the verb. Other terms, as used in the present invention, shall be defined by their well-known meanings in the art.

[0189] It is recognized that certain features of the invention, which are, for greater clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, several features of the invention, which are, for greater brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as appropriate in any other described embodiment of the invention. Certain features described in the context of several embodiments should not be considered essential features of those embodiments unless the embodiment is inoperable without those elements.

[0190] All publications, patents and patent applications mentioned in this descriptive report are incorporated into the present invention in their entirety by reference to the descriptive report, to the same extent as if each individual publication, patent or patent application were specifically and individually indicated for incorporation into the present invention by reference. Furthermore, the citation or identification of any reference in this descriptive report is prohibited. Petition 870250101549, dated 06 / 11 / 2025, pp. 73 / 104 Application 70 / 92 should not be interpreted as an admission that such reference is available as a prior art to the present invention. To the extent that section headings are used, they should not be interpreted as necessarily limiting. EXAMPLES

[0191] In general, the nomenclature used in the present invention and the laboratory procedures used in the present invention include molecular, biochemical, bioengineering, bioprocessing, microbiological, and recombinant DNA techniques. Such techniques are exhaustively explained in the literature. See, for example, “Molecular Cloning: A Laboratory Manual” Sambrook et al., 1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York 1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Handbook Series”, Volumes 1-4, Cold Spring Harbor Laboratory Press, New York 1998); methodologies as set forth in U.S. Patents Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, volumes I-III, Cellis, JE, ed. 1994); “Animal Cell Culture - A Basic Technique Manual” by Freshney, Wiley-Liss, NY 1994), third edition; “Current Protocols in Immunology” volumes I-III, Coligan JE, ed. 1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th edition), Appleton & Lange, Petition 870250101549, dated 06 / 11 / 2025, pp. 74 / 104 71 / 92 Norwalk, CT (1994); Mishell e Shiigi (orgs.), “Selected Methods in Cellular Immunology, W. H. Freeman and Co., Nova York (1980); Molecular Cell Biology Berk A. et al. 8aedição; Molecular Biotechnology : Principles and Applications of Recombinant DN, Glick BR. 5a edição; Culture of Animal Cells : A Manual of Basic Technique and Specialized Applications Freshney IR, 7a edição; “Oligonucleotide Synthesis Gait, M. J., ed. (1984); “Nucleic Acid Hybridization Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation Hames, B. D., and Higgins S. J., eds. L1984); “Animal Cell Culture Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes IRL Press, (1986); “A Practical Guide to Molecular Cloning Perbal, B., L1984) e “Methods in Enzymology Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications, Academic Press, São Diego, CA (1990); Marshak et al., “Strategies for Protein Purification and Characterization - A Laboratory Course Manual CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document. EXAMPLE 1 Bioinformatics analysis for the identification of new genomic refuge sites.

[0192] In order to identify genomic locations on the chicken Z chromosome that could be targeted by a bioreporter expression cassette, such as a gene encoding red fluorescent protein, the inventors performed a comprehensive bioinformatics analysis, applying firm criteria for the allocation of potentially safe new genomic refuge sites. Petition 870250101549, dated 06 / 11 / 2025, pp. 75 / 104 72 / 92

[0193] The inventors based their bioinformatics analysis on criteria used in previously published work aimed at identifying safe genomic refuge sites in other genomes. Differences in genome size and chromosome number were taken into account in the analysis (e.g., 39 chromosomes containing ~1.2 Gb for the chicken genome, compared to 20 chromosomes containing ~2.5 Gb and 23 chromosomes containing ~3 Gb in the mouse and human genomes, respectively). The following criteria were defined for the identification of safe harbor genomic sites: • Criterion 1 - the distance from the genomic site to the end of any gene must be greater than 20 kb. • Criterion 2 - the distance from the genomic site of any cancer-related gene must be greater than 120 kb. • Criterion 3 - the distance from the genomic site of any microRNA (miRNA) must be greater than 120 kb. • Criterion 4 - the genomic site must be located outside of CpG islands, which generally represent transcription units such as topological association domain(s), TAD(s), enhancers, or promoters. • Criterion 5 - the genomic site must be located outside of ultraconserved genomic regions. • Criterion 6 - the genomic site must exclude duplicate elements.

[0194] The inventors performed the bioinformatics analysis using the platform offered by the genome browser at the University of California, Santa Cruz (UCSC). Petition 870250101549, dated 06 / 11 / 2025, pp. 76 / 104 73 / 92

[0195] Genomic data, assembly, mapping and sequencing: • UCSC Genome Browser assembly ID: galGal6 • Sequencing / assembly provider ID: GRCg6a Genome Reference Consortium • Assembly date: March 2018 • Accession ID: GCF_000002315.5 • NCBI Genome ID: 111 (Gallus gallus) • NCBI Assembly ID: 1668981 • NCBI BioProject ID: 13342 • NCBI Biological sample ID: SAMN02981218

[0196] Genes and gene predictions: • TransMap Alignments Version 5 (Data last updated at UCSC: 06 / 10 / 2019) • TransMap Ensembl and GENCODE Version 5 Mappings • Ensembl Gene Predictions, Source Data Version: 104 (Data last updated at UCSC: 05 / 25 / 2021) • NCBI RefSeq Gene Predictions - NCBI Gallus Release Annotation Gallus Release Annotation 104 (Data last updated at UCSC: 03 / 29 / 2020) • Non-Chicken RefSeq Genes • GenBank Chicken mRNAs • GenBank Non-Chicken mRNAs • UnlProt SwlssProt / TrEMBL Protein Annotations (Source Data Version at UCSC: UnlProt Knowledgebase Release 2020_05)

[0197] miRNA predictions: • miRBase Petition 870250101549, dated 06 / 11 / 2025, pp. 77 / 104 74 / 92 • MiRscan [ 0198]Expression and Regulation: • Tracks of CpG Islands (Islands < 300 bp)

[0199] Comparative Genomics: • Multizone Alignment and Conservation of Vertebrates (multiple alignments of all 77 vertebrate species) • Bird Chain and Network Alignments

[0200] Variation and Repetitions: • Repeatable Elements by RepeatMasker (Intercalated short nuclear elements, including ALUs, Intercalated long nuclear elements, Long terminal repeat elements, including retroposons, Satellites, microsatellites, Low complexity repeats, RNA repeats).

[0201] The inventors successfully identified 18 potential genomic locations on the Z-gender chromosome through their unbiased bioinformatic screening, which met the rigorous criteria applied. Table 1. Initial screening of safe harbor loci on the Z-gender chromosome, applying criteria #1 to #6. The loci are in accordance with or based on the UCSC Genome Browser assembly ID: galGal6. No. of Location Genomic location (number of nucleotides on chromosome Z) Site 1 chrZ: 1,550,000 - 1,730,000 Site 2 chrZ: 4,075,000 - 6,150,000 Site 3 chrZ: 17,100,000 - 17,450,000 Site 4 chrZ: 17,550,000 - 18,050,000 Petition 870250101549, dated 06 / 11 / 2025, pp. 78 / 104 75 / 92 Site 5 chrZ: 19,500,000 - 20,000,000 Site 6 chrZ: 26,000,000 - 26,400,000 Site 7 chrZ: 30,000,000 - 30,800,000 Site 8 chrZ: 36,000,000 - 36,600,000 Site 9 chrZ: 38,500,000 - 39,200,000 Site 10 chrZ: 48,300,000 - 50,000,000 Site 11 chrZ: 50,900,000 - 51,400,000 Site 12 chrZ:51,425,000 - 51,625,000 Site 13 chrZ:58,500,000 - 59,700,000 Site 14 chrZ:61,400,000 - 62,400,000 Site 15 chrZ:63,150,000 - 63,550,000 Site 16 chrZ:64,350,000 - 65,250,000 Site 17 chrZ:697,000,000 - 70,400,000 Site 18 chrZ:73,500,000 - 78,800,000 Table 2. Size and additional characteristics of genomic sites no. 1-18 No. Location Size Comments miRNA (Y or N) Comments Site 1 165 Kb 2 human mirRNAs, neighbors of Smad2 N Site 2 2 Mb human mirRNAs, multiple loci N Site 3 2 60 Kb One human lncRNA, one mouse gene N Petition 870250101549, dated 06 / 11 / 2025, pp. 79 / 104 76 / 92 Site 4 4 ​​60 Kb Few very small N loci Site 5 42 0Kb OK Nearest N mirRNA: 18874996 (875 Kb) Site 6 2 90 Kb Several N loci Nearest N mirRNA: 27510654 (1 Mb) Site 7 7 00 Kb One small gene in the middle N Site 8 480 Kb Few very small N loci Nearest N mirRNA: 35181264 (800 Kb) Site 9 600 Kb Several loci, close to the centromere Nearest N mirRNA: 40078117 (800 Kb) Site 10 1.5 Mb One human gene in the middle Y mirRNA at 48684570 Site 11 450 Kb Several loci Meleag, near the CHD1Z gene, N Site 12 185 Kb One long Meleag locus N Site 13 800 Kb Multiple loci Ensemble and nearest N mirRNA: Petition 870250101549, dated 06 / 11 / 2025, pp. 80 / 104 77 / 92 Predictions. 60469242 (900 KB) Site 14 900 KB Two Cuteron loci on the side. Two small loci in the middle. N 2 nearby mirRNAs: (500 Kb) and (450 Kb) Site 15 350 Kb One human locus in the middle N nearest mirRNA: 64304208 (900 Kb) Site 16 800 Kb Multiple Ensembke and small loci Y mirRNA at 64670256; nearby mirRNA (40 KB); Site 17 700 Kb Some Cuteron loci, near Jae Han N insertion site Site 18 5.1 Mb Multiple LOC N set and predictions Near mirRNA at 78512252

[0202] Next, genomic sites #1 to #18 met the following criteria, so the inventors filtered out non-cytogenetic genomic sites: Petition 870250101549, dated 06 / 11 / 2025, pp. 81 / 104 78 / 92 • Criteria 7 - the sites must be located outside the centromere of the Z gender chromosome (~chrZ:42,150,000 - 42,260,000). • Criterion 8 - the sites must be located outside the ZW asynapsis, the sites with a potential risk of recombination between the Z and W gender chromosomes.

[0203] After subjecting genomic sites #1 to #18 to criteria #7 and #8, the inventors narrowed their list down to 10 loci on the Z-gender chromosome, as described in Table 3. First, of the 18 potential sites of origin, only the genomic loci within sites #3, 4, 5, 7, 8, 13, 14, and 15 met the cytogenetic-based criteria. Furthermore, by applying these cytogenetic-based criteria, the size of each genomic site that met these criteria was reduced. The new reduced safe harbor loci, which met criteria #1 to #8, were named after the site of origin number with the addition of a small last (e.g., site 3a is the reduced genomic site of site 3). As shown in Table 3, regarding sites #7 and #14, two different potential safe harbor loci were found for each site, designated sites #7a, 7b, 14a, and 14b, respectively. Table 3. Safe harbor loci on the Z chromosome after cytogenetic criteria. No. of Location Genomic location (number of nucleotides on chromosome Z) Site 3a chrZ: 17,141,000 - 17,406,000 Site 4a chrZ: 17,735,000 - 18,020,000 Petition 870250101549, dated 06 / 11 / 2025, pages 82 / 104 79 / 92 Site 5a chrZ: 19,560,000 - 19,995,000 Site 7a chrZ: 30,060,000 - 30,270,000 Site 7b chrZ: 30,465,000 - 30,750,000 Site 8a chrZ: 36,215,000 - 36,545,000 Site 13a chrZ: 59,100,000 - 59,430,000 Site 14a chrZ: 61,530,000 - 61,865,000 Site 14b chrZ: 61,875,000 - 62,315,000 Site 15a chrZ:63.210.000 - 63.440.000

[0204] Next, the inventors ranked the ten sites described in Table 3 according to an internal scoring system based on all their findings. The four highest-ranked sites were: site 4a, site 5a, site 7b, and site 14a, as described in Table 4. Table 4. Best-ranked sites on the Z-genus chromosome for insertion of a bioreporter expression cassette. Location No. Genomic Location Genomic Region Size Site 4a chrZ: 17,735,000 - 18,020,000 2 85 Kb Site 5a chrZ: 19,560,000 - 19,995,000 435 Kb Site 7b chrZ: 30,465,000 - 30,750,000 2 85 Kb Site 14a chrZ: 61,530,000 - 61,865,000 335 Kb Petition 870250101549, dated 06 / 11 / 2025, pp. 83 / 104 80 / 92

[0205] Next, the inventors designed multiple gRNAs corresponding to sites n°s: 4a, 5a, 7b and 14a, to assess their accessibility to Cas9 and the potential for gene editing and knock-in (Kl) at these sites. The gRNAs were designed using the CCTop CRISPR / Cas9 online target predictor.

[0206] gRNAs were designed according to the following parameters: • Cas9: Streptococcus pyogenes Cas9 (a single gRNA per site) • Protospacer adjacent motif (PAM) site: NGG • Genome target: Gallus gallus, Ensemble V103 • Core length = 12 bp • Protospacer length = 20 bp • Off-target score: CRISPRater score > 0.74 • Maximum number of off-target nuclear mismatches = 2 • Off-target search method: comprehensive and exhaustive across the entire genome.

[0207] Among all the gRNAs examined, three gRNA molecules, named gRNA6.1 (SEQ ID NO: 1), gRNA7.2 (SEQ ID NO: 2), and gRNA9.1 (SEQ ID NO: 3), were considered the most suitable for DNA cleavage at sites 4a, 5a, and 14a, respectively. The sequences of these gRNAs are described in Table 5 below. Thus, from the original 18 potential sites, three final genomic sites within the Z genus chromosome were selected, with Petition 870250101549, dated 06 / 11 / 2025, pp. 84 / 104 81 / 92 three corresponding gRNA molecules for further evaluation. Table 5. Three main localization sites and their corresponding gRNA molecules. gRNA Location No. (SEQ ID NO:) gRNA Sequence Filament CRISPR Score Site 4a gRNA6.1 (SEQ ID NO: 1) ACATGCAATACACTGAAC TG + 1 0.80 Site 5a gRNA7.2 (SEQ ID NO: 2) ACTGTTAACAAGGTTGGT TG + 1 0.88 Site 14a gRNA9.1 (SEQ ID NO: 3) CTTGTAGGGCTTGATTAC TG + 1 0.85

[0208] The next objective was to evaluate the potential efficiency of gene editing at the three highest-ranked sites above, with their corresponding gRNA molecules (Table 5). DF1 cells, a chicken embryo fibroblast cell line, were separately electroporated with Cas9 / guide RNA (gRNA) and ribonucleoprotein (RNP) complexes to test the efficiency of DNA cleavage at the highest-ranked sites: site 4a, site 5a, and site 14a, with the gRNA molecule. Petition 870250101549, dated 06 / 11 / 2025, pages 85 / 104 82 / 92 corresponding. The DNA samples were tested using the T7 endonuclease I mismatch cleavage assay (T7EI), which determines genome editing at the target and provides an estimate of genome editing efficiency in CRISPR-treated cells.

[0209] As demonstrated in Figure 1, according to the T7E1 assay, 85.1%, 41.1%, and 45.8% of the dsDNA molecules were cleaved by the Cas9 / gRNA RNP complexes, comprising gRNA6.1, gRNA7.2, and gRNA9.1, respectively, thus indicating a relatively high potential for these loci as suitable for the insertion of the exogenous RFP reporter gene.

[0210] Next, the DNA samples were subjected to Sanger sequencing, followed by TIDE analyses to track DNA insertions and deletions (indels). As observed in Figure 2, the highest percentage of indels was observed for the examined gRNA molecules: gRNA6.1 (SEQ ID NO: 1), gRNA7.2 (SEQ ID NO: 2), and gRNA9.1 (SEQ ID NO: 3), compared to other candidate gRNA molecules.

[0211] Therefore, the inventors conclude that, after the narrowing process disclosed in the present invention, specific, improved and non-trivial protection sites were developed for the proper integration of a nucleic acid sequence of an exogenous reporter gene encoding RFP on the Z sex chromosome of a chicken. EXAMPLE 2 Molecular and functional characterization of genome-edited chicken PGCs

[0212] To ensure the successful generation of at least three DsRed-positive PGCs, crucial for the subsequent Petition 870250101549, dated 06 / 11 / 2025, pages 86 / 104 In the 83 / 92 study of chimeric rooster breeding and a detectable sexually mature flock, the inventors focused on five SHLs. Selection criteria included culture viability, proliferation rates, and fluorescence stability. After careful evaluation, the inventors prioritized three SHL sites that demonstrated the most promising results.

[0213] The five SHLs initially targeted are specified in Table 6. Table 6. No. of Location Genomic location (number of nucleotides on chromosome Z) Site 4a chrZ: 17,735,000 - 18,020,000 Site 7b chrZ: 30,465,000 - 30,750,000 Site 8a chrZ: 36,215,000 - 36,545,000 Site 13a chrZ: 59,100,000 - 59,430,000 Site 14a chrZ: 61,530,000 - 61,865,000

[0214] Generally, the workflow for each site encompassed five phases: (1) Evaluation of several guide RNAs (gRNAs) targeting the genomic site to facilitate DNA cleavage by Cas9; (2) Construction of two plasmids per site: (i) CRISPR / Cas9 plasmid containing spCas9 and the selected site-specific gRNA, and (ii) DsRed HDR cassette flanked by site-specific homology arms; (3) Introduction of the plasmids into PGC derived from a Lohmann-LSL male chicken embryo; (4) Implementation of sorting procedures to enrich DsRed-positive cells; and (5) Molecular validation to confirm the accurate integration of DsRed into each of the desired SHLs. Petition 870250101549, dated 06 / 11 / 2025, pages 87 / 104 84 / 92 Results Finding the right gRNA for each target genomic site

[0215] For each of the five genomic SHLs, the inventors used the online tool CCTop to design gRNAs with the following specifications: - Cas9: Streptococcus pyogenes Cas9 (a single gRNA per site) - Protospacer adjacent motif (PAM) site: NGG - Genome target: Gallus gallus, Ensemble V103 - Core length = 12 bp - Protospacer length = 20 bp Off-target score: CRISPRater score >0.74. Maximum out-of-target core mismatch = 2 - Off-target search method: comprehensive and exhaustive across the entire genome.

[0216] The inventors designed 2 to 4 gRNAs for each SHL, except for sites 4a and 14a, for which the inventors had already designed high-quality gRNAs.

[0217] Each gRNA was tested as a ribonucleoprotein (RNP) complex together with Cas9 in the DF1 chicken cell line using electroporation.

[0218] DF1 cells were collected 2 to 3 days after electroporation, and DNA was extracted to assess cleavage using a T7 assay.

[0219] The T7 products were analyzed on agarose gel, compared to non-transfected and non-cleaved control groups (Figure 3) and subsequently subjected to sequencing for cleavage quantification using the TIDE online analysis tool. Petition 870250101549, dated 06 / 11 / 2025, pages 88 / 104 85 / 92

[0220] gRNAs that demonstrated efficient cleavage rates in both gel electrophoresis and TIDE analysis were selected for further steps (Tables 7-8). Table 7. Quantification of cleavage facilitated by newly designed gRNAs using TIDE analysis. gRNA TIDE quantification * 7b.1 20% 7b.2 13% 8a.1 12% 8a.2 2% 8a.3 2% 8a.4 6% 13a.1 7% 13a.2 55% 13a.3 45% 13a.4 40% * When applicable, the average T7 quantification was calculated using forward and reverse sequencing data. Table 8. gRNAs selected for each SHL based on gel electrophoresis and TIDE quantification. SHL gRNA Sequence SEQ ID NO: 4a 6.1 ACATGCAATACACTGAACTG 1 7b 7b.1 TCTTGCTTTCCACTTTCCAT 18 14a 9.1 CTTGTAGGGCTTGATTACTG 3 Petition 870250101549, dated 06 / 11 / 2025, pp. 89 / 104 86 / 92 8a 8a.4 AAGTGTGGAACAAACTGCTG 19 13a 13a.2 GATGGGCTCCACAAGGAACT 20 Plasmid cloning

[0221] Based on the selected gRNA sequences, two plasmids were created for each genomic site. Ll) CRISPR / Cas9 plasmid

[0222] This plasmid harbors spCas9 along with site-specific gRNA under a human U6 promoter. In addition, EGFP acts as a reporter gene to assess transfection efficacy.

[0223] The plasmids were sequencing to verify the precise sequences of critical segments, including the Cas9 promoter and ORF, as well as the U6 promoter and gRNA sequence. For sites 4a and 14a, sequencing revealed mutations in these regions. Consequently, the inventors used plasmids from other sites, exhibiting correct sequences, for gRNA cloning targeting sites 4a and 14a. In these cases, the gRNA promoter was obtained from chicken U6. The resulting cloning products were validated by Sanger sequencing. (2) DsRed HDR Plasmid

[0224] This plasmid contains the DsRed cassette, comprising the CBh promoter, the ORF, and the PolyA signal of SV40. The cassette is flanked by 250 bp site-specific homology arms, facilitating integration into the cleaved region of chromosome Z (Table 9). All cloned plasmids were subjected to validation by Sanger sequencing. Petition 870250101549, dated 06 / 11 / 2025, pages 90 / 104 87 / 92 Table 9. Homologation arms flanking the cassette expressing DsRed SHL homology arm sequence 5' homology arm sequence 3' 4a GGCCTATACAGACAAATCTTTACTAAA TGGAGAATCCTGACATTTTGTCCATTT CTCTCTATACCACCAACTCTCATCCTTG GTTACATTGCCTGTCAGTTGAACAGCCATCCATCCATCCATTCAACCATTCACATTTGTCCTATACCACCAACTCATCCATTCATTCATTT TTATCTTGAATTGAGCCATCAAGAGGT ATTTCTCTGATACACCATTATCTAAAA CCCAGTGTTATTTTTCCAGTGAGGGTAG ATTCCCTTTACTGCTTTTTCTCCTCAT TTCCA (SEQ ID NO: 12) ATAACGGCATTCAGTACTTGTCAT CACTAATTAGTATTACCATCCATCCATCTGACTGTTATGTTAT TTAATAATTCCACCATAAATTAGTT CTATGAGCATTTGATCTAGTGGT TGTTGTAGATCACAGGATATGAAT AGATTGTGTGTGTGTGTAAACCAG GAGTGTCTTGACAAAAGGACAG TGCTGGTAAGACCAGCAGAAGAGG GTCCCCTGTGGCAAAAATGTTTTA CTGACCT 13. IDTTG: NOTE: NOTE: TGAATGTGTGGAAAGTGAAGATAACTACT AGTGCAGCAAGCAGACATAGAGATTTT ATAAAAGTGGATGGTGAGCATTGTTTG TTAGATACAAACCATTAAGCAGAGATG CCTAGTGACTCAGAGTACAGAGCTTCC TGACTGTCAACACTGACCATGACACTG ACCACGTTATGAGAGAGATTAGACCTTTATTTATTTATTTA AATTGTAATCACAGAAGAGGACAAACA GTGATAG (SEQ ID NO: 21) TCTATTGTCTCATAAGTCCTACCA CTCAGGAATAAGGCTTATAAGAAA CATGAGAAATGTGTAACTGTAGAGTGAGTTAGACATAGCCAGCAGCTA TATACGTACCCATATGCTCACAGT CTCTCTCCTTCGCAGGATTAGGAA ATAAAATTGGGTGAAAAAGGTTAT AGATAAGACAGAGATAGAAAGATT GCTAATCATTTAACATATTTGATT TGGGCAAAGTAATTGAATACACTG TCAAGTAAG (22) ID: 22 NO GGTCTGTGCAAACAGTGTTTCTCATGC AACTTGGCTGCCTTTAAATGGCTTACC ACATCACCAGGGCTGGAGGTTACA TCCTGCTGGCCAAAACTACATTAT Petition 870250101549, of 06 / 11 / 2025, p. 91 / 104 88 / 92 AACTCTTTTCTGAAAAACCTAAAAAAT GTCTGTGTGCCAAGTAAGATACTTCAA TTCAAAAGGAGGTTTTTCAATTTTTTC TCTCAGTTTATCATTTCTTCTACTTGA AAAATATATTTTAATTTTAATGTTTTT TGTTTTATACAAAAATATATGAACTAT GTATTACTATTGTCACCTGTCATTACC AAGAAGT (SEQ ID NO: 16) GGTGGGTTAGCTAATTTTACAGGA GGGATAAATTGTGAGAATCTGGAA ACCATAATCTTGACAAGAAAAAAT TAACACCCAATTCCTTGGTGAGAC TGGGCATTATTGGACATGGGAAA TCTCACAGTCATGATATATGTTAG GAAGGAACTCTCCTTCAAGGGAGCCTAGCCTAGCCTAGCCTT CAACACTTGG (SEQ ID NO: 17) 8a CAAGAACATTAAAAAGAAACAGGATCT CTCTTGCTTTTGTTAAGAAAAAAAAAC AGCAGCAGTACACCAAGCAGTTAAA TTACTCTATTTAGATGTCTGAAACA CTTCTAAAGCAAAGCAGGTAATACTTC AGGGACCATCTACTTCATTCATTTCTTTCTTTCTTTCATTCATTAGCTGAAACA GATCTTTCTGTGGAAAAATACAAAATT GTGTCTGTTTCTTGAAGACCGGTAACC AGAAAGT (SEQ ID NO: 23) TATTTGTCAGAAAAATCTATCTGC ATACTCATTTCTTTGAATGATT ACAATCATGAGATGTCCACTATCT GCATTTTTGATCACTAGTGAATTAGTCCACTCATTCAGTCCACTGATT ATTATTGTTTATTTAGGAGCAG GCCTTTAAAAGAAATACTGTGGTC ACCTGTGTGACTGAAAAAAGCACA TATTCAATTCATGTGAATAACATT AGAAAGTTTCCAGGAACTGCTGAT GATTCAAGCA (SEQ ID NO:24). TGACAGAGCAGTGCTTACAGAGTCAAA TGCTTTGCTTGCACATGCAGTAAA CtttTTTAATCTTAATTCATAAAC TCTCACACTTTTACATTTCCAATT TTCACATCTTTAATCATCTGGGAG ATGATTTCACATCCTCATCAAGTT TGTGGATGACCCAGAACTGAGGGCTAGCTCGAGCGGAGGGAG TGCTGCCATCTGGAGGAACTTGCA Petition 870250101549, of 06 / 11 / 2025, p. 92 / 104 89 / 92 AACTTTTTATTTTATTGTGCTATCCTG CCAATGA (SEQ ID NO: 25) CAGTTGGAGAAGGGCTGACAGGAA CTTGATGGAGTTCTACATGGAGAA GGGAAAGTTC (SEQ ID NO: 26) Insertion of plasmids into male-derived Lohmann-LSL PGCs

[0225] To facilitate integration of the DsRed gene into the desired SHL on the Z chromosome, PGCs were cotransfected with CRISPR / Cas9 and DsRed HDR plasmids.

[0226] The CRISPR / Cas9 plasmid induces a double-strand break at the target genomic locus, while the DsRed plasmid facilitates repair by integrating the DsRed cassette into the cleaved site.

[0227] Two to three (2-3) days after transfection, fluorescent signals of GFP and DsRed were observed in PGCs (Figure 4). These signals showed distinct green and red fluorescence, with numerous cells being positive for both signals. Screening procedures for enriching DsRed-positive cells

[0228] To isolate cells exhibiting stable expression of the fluorescent protein DsRed, the red blood cell population was subjected to multiple rounds of screening procedures. For each specific target genomic region, cells were screened approximately 4 times, every 1-2 weeks, until a consistently stable red PGC culture was achieved (Figure 5).

[0229] Among the five target SHLs, three demonstrated robust and consistent fluorescence in their cultures, Petition 870250101549, dated 06 / 11 / 2025, pp. 93 / 104 90 / 92 along with sustained proliferation: 4a, 14a and 13a. These three stable PGC DsRed+ cultures were subsequently expanded. Molecular validation of the correct integration of DsRed into the desired SHL.

[0230] To validate the accurate integration of the DsRed cassette into the Z chromosome SHL of the three stable PGC cultures, two PCR assays were conducted with primers flanking the 5' or 3' homology arms of the cassette (Table 10). Table 10. PCR primers to validate the integration of the DsRed cassette into an SHL. T Region Forward Primer Reverse Primer 4a 5' CACAGGGAGATTTGACAGTG G (SEQ ID NO: 27) GGGCGTACTTGGCATATGAT (SEQ ID NO: 28) 3' CCCCCTGAACCTGAAACATA (SEQ ID NO: 29) GAGACTTTTGTAGCTTGACTTG G (SEQ ID NO: 30) 13a (Se t #1) 5' CAGATCTTGGTTGAGATGCA TC (SEQ ID NO: 31) AAGTGGGCAGTTTACCGTAAAT AC (SEQ ID NO: 32) 3' CCCCCTGAACCTGAAACATA (SEQ ID NO: 33) GTTCTAAGGGAAGGACCACGT (SEQ ID NO: 34) 13a (Se t #2) 5' CAGATCTTGGTTGAGATGCA TC (SEQ ID NO: 35) GGGCGTACTTGGCATATGAT (SEQ ID NO: 36) 3' GCATTCTAGTTGTGGTTTGT CC (SEQ ID NO: 37) GTTCTAAGGGAAGGACCACGT (SEQ ID NO: 38)

[0231] PCR analyses produced the expected product sizes (Figure 6), confirming the amplification Petition 870250101549, dated 06 / 11 / 2025, pp. 94 / 104 91 / 92 successful targeting of the target sequences. Sanger sequencing further validated the expected sequence integration within the SHL. EXAMPLE 3 In ovo injection of PGC to validate its fluorescent characteristics.

[0232] To demonstrate that genome-integrated DsRed cells (e.g., DsRed-positive PGCs), as disclosed in the present invention, are detectable through the shell of an egg, infertile eggs were injected with control PGCs (e.g., unedited PGCs) or gene-edited PGCs containing the DsRed gene. The number of cells injected was equivalent to the number of cells in an egg embryo on the third embryonic day.

[0233] The results show that no fluorescent signal was detected by injection of unedited parental control PGC (Figure 7B). In sharp contrast, a distinct fluorescent focus was observed after injection of DsRed-positive chicken PGCs.

[0234] Thus, it is concluded that chicken PGCs comprising the DsRed coding sequence integrated into a safe harbor site in the PGC genome were successfully produced. A practitioner with common knowledge of the art would recognize that fully grown chickens can be obtained from the disclosed PGCs, according to methods known in the art.

[0235] Although the present invention has been described in detail, professionals skilled in the art will recognize that many variations and modifications can be made. Therefore, the invention should not be Petition 870250101549, dated 06 / 11 / 2025, pp. 95 / 104 92 / 92 interpreted as restricted to the embodiments specifically described, and the scope and concept of the invention will be more easily understood by reference to the following claims. Petition 870250101549, dated 06 / 11 / 2025, pp. 96 / 104

Claims

1 / 5 CLAIMS 1. Transgenic female chicken, CHARACTERIZED IN THAT it comprises a recombinant gender Z chromosome comprising a nucleic acid sequence of an exogenous reporter gene encoding a red fluorescent protein (RFP), at least one location on a gender Z chromosome of a female chicken, wherein said at least one location is listed in Table 1.

2. Transgenic female chicken, according to claim 1, CHARACTERIZED IN THAT said at least one location is selected from the group consisting of: site 3, site 4, site 5, site 7, site 8, site 13, site 14, site 15, and any combination thereof.

3. Transgenic female chicken, according to claim 1 or 2, CHARACTERIZED IN THAT at least one of these locations is listed in Table 3.

4. Transgenic female chicken, according to claim 3, CHARACTERIZED IN THAT said at least one location is selected from the group consisting of: site 4a, site 5a, site 7b, site 13a, site 14a, and any combination thereof.

5. Transgenic female chicken, according to claim 4, CHARACTERIZED IN THAT said at least one location is selected from the group consisting of: said site 4a, said site 13a, said site 14a, and any combination thereof.

6. Transgenic female chicken, according to any of claims 1 to 5, CHARACTERIZED BY Petition 870250101540, dated 06 / 11 / 2025, p. 5 / 20 2 / 5 FACT THAT said RFP is an excitation wavelength of 500 to 650 nm and an emission wavelength of 550 to 650 nm.

7. Cell, CHARACTERIZED by the fact that it is obtained from or derived from a transgenic female chicken, according to any one of claims 1 to 6.

8. Cell according to claim 7, characterized in that it is a primordial germ cell (PGC).

9. Method for determining the sex of a chicken embryo in an unhatched egg comprising the embryo within a structurally intact shell, the method CHARACTERIZED BY THE FACT that it comprises: (a) obtaining at least one unhatched egg comprising an embryo within a structurally intact shell, from the transgenic female chicken according to any one of claims 1 to 6; and (b) determining whether a red fluorescent signal is detected in said embryo residing in said unhatched egg, wherein the detection of said red fluorescent signal indicates the expression of said RFP in said embryo within said structurally intact shell of said unhatched egg and thus indicates the presence of said recombinant Z chromosome in said embryo, thereby determining that said chicken embryo in said unhatched egg is a male embryo.

10. Method, according to claim 9, CHARACTERIZED BY THE FACT THAT the detection of the absence of red fluorescent signal in said embryo residing in said unhatched egg indicates that RFP is not expressed in said embryo and thus indicates the absence of said recombinant Z chromosome in said embryo, thus determining that said chicken embryo in said unhatched egg is a female embryo.

11. Method, according to claim 9 or 10, CHARACTERIZED IN THAT it further comprises a step comprising subjecting said unhatched egg comprising the embryo within a structurally intact shell to a light source.

12. Method, according to any one of claims 9 to 11, CHARACTERIZED IN THAT it further comprises a step prior to said step (b) comprising isolating at least one female embryo from said transgenic female chicken.

13. Kit, characterized in that it comprises at least one first nucleic acid molecule encoding a guide RNA (gRNA), comprising the nucleotide sequence as presented in any of the SEQ ID Nos: 1 - 3, and 18 - 20.

14. Kit, according to claim 13, CHARACTERIZED IN THAT it additionally comprises at least one second nucleic acid molecule encoding any one of: a clustered regularly interspersed short palindromic repeats (CRISPR) associated protein 9, an RFP, or both.

15. Kit, according to claim 14, CHARACTERIZED IN THAT it further comprises instructions for integrating said at least second nucleic acid molecule encoding RFP into at least one Z chromosome location of a female chicken, Petition 870250101540, dated 06 / 11 / 2025, page 7 / 20 4 / 5 wherein said at least one location is listed in Table 1.

16. Method for producing a chicken comprising a recombinant gender Z chromosome, the method CHARACTERIZED BY THE FACT THAT it comprises: (a) obtaining at least one transgenic PGC comprising a recombinant Z chromosome comprising a nucleic acid sequence of an exogenous reporter gene encoding an RFP at least one location as listed in Table 1 on a chicken's gender Z chromosome; and, (b) transplanting said transgenic PGC into a receptive chicken embryo, thereby producing a chicken comprising the recombinant gender Z chromosome.

17. Method according to claim 16, CHARACTERIZED IN THAT it further comprises a step prior to step (a), comprising integrating said nucleic acid sequence of an exogenous reporter gene encoding an RFP at least one location as listed in Table 1 on the gender Z chromosome of a PGC, thereby obtaining said transgenic PGC.

18. Method according to claim 16 or 17, characterized in that at least one location said is listed in Table 3.

19. Method, according to any one of claims 16 to 18, CHARACTERIZED IN THAT said at least one location is selected from the group consisting of: site 4a, site 13a, site 14a, and any combination thereof. Petition 870250101540, dated 06 / 11 / 2025, page 8 / 20 5 / 5 20. Method, according to any one of claims 16 to 19, CHARACTERIZED in that said nucleic acid sequence of an exogenous reporter gene encoding an RFP is integrated into said location of said gender Z chromosome using a type II CRISPR system, comprising Cas9 protein and a gRNA.

21. Method according to claim 20, CHARACTERIZED in that said gRNA comprises the nucleotide sequence presented in any of the SEQ ID Nos: 1, 3, and 20.

22. Method according to claim 21, CHARACTERIZED in that said nucleic acid sequence of an exogenous reporter gene is integrated into said site 4a, said site 13a, or said site 14a, using said Cas9 protein and a gRNA comprising the nucleotide sequence as presented in SEQ ID NO: 1, SEQ ID NO: 20, or SEQ ID NO: 3, respectively. Petition 870250101540, dated 06 / 11 / 2025, p. 9 / 20

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