Mettl7a for improved embryo competence

Abstract

Provided herein are methods for improving in vitro produced embryo competence by increasing METTL7A. Also provided are compositions comprising METTL7A or nucleic acids encoding METTL7A for use in such methods.

Description

METTL7A FOR IMPROVED EMBRYO COMPETENCEGOVERNMENT INTERESTS

[0001] This invention was made with government support under Grant No. R01 HD102533, awarded by the National Institutes of Health; and Grant No. 2019-67016-29863, awarded by the US Department of Agriculture, National Institute of Food & Agriculture. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Application No. 63 / 698,174, filed September 24, 2024, which is herein incorporated by reference in its entirety for all purposes.REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN XML FILE

[0003] The Sequence Listing written in file 634762SEQLIST.xml is 20,480 bytes, was created on July 23, 2025, and is hereby incorporated by reference.BACKGROUND

[0004] In vitro fertilization (IVF) is currently one of the most used assisted reproductive technologies to achieve a successful pregnancy, both in humans and a number of agricultural species. However, the competence of in vitro produced embryos to establish pregnancy is much lower than the embryos produced in vivo. It is believed these complications associated with IVF embryos are induced by the environmental stressors accumulated during in vitro embryo culture.

[0005] Gametes and embryos are exposed to high levels of oxidative stress in in vitro culture conditions. Oxygen (O2) tension is one of the major sources that lead to oxidative stress. The atmospheric concentration of O2 (20%) used in the embryo culture system is considerably greater than the oxygen tension in the oviduct and uterus of mammals. Studies across multiple species have shown improved in vitro embryo development when oxygen levels are reduced from 20% to 5%. This elevated O2 level during embryonic development can influence gene expression, metabolism, and the activity of important epigenetic enzymes. Another major contributor of oxidative stress is reactive oxygen species (ROS), which are by-products of oxidativephosphorylation within the mitochondria. Under physiological conditions, ROS level is closely monitored and controlled by antioxidants. However, mitochondrial dysfunction and impaired antioxidant defense system can lead to the generation of excess ROS, resulting in delayed development, DNA damage, apoptosis, or lipid peroxidation.

[0006] To mitigate the detrimental effect of oxidative stress on in vitro embryo culture and promote embryo competence, multiple approaches have been explored beyond reduced oxygen tension. Such additional measures include co-culture with cumulus cells as ROS scavenger, or supplementation of exogenous antioxidants to reduce ROS (e.g., anethole, beta-mercaptoethanol, imperatorin, N-(2-mercaptopropionyl)-glycine, and dihydromyricetin). Boosting endogenous antioxidants such as glutathione (GSH in reduced form) has also been tested. However, none of these methods have fully overcome the competence issues associated with in vitro produced embryos. Accordingly, a need remains to improve in vitro produced embryo competence.SUMMARY

[0007] Provided herein are methods for improving in vitro produced embryo competence comprising increasing METTL7A in an embryo. In some such methods, exogenous METTL7A protein or a fragment thereof is introduced into the embryo. Optionally, the introducing comprises microinjection into a presumptive zygote or one blastomere of a two-cell embryo. In some such methods, a polynucleotide encoding METTL7A is introduced into the embryo. Optionally, the polynucleotide is DNA. Optionally, the polynucleotide is RNA.

[0008] In some such methods, the introducing comprises: (i) microinjection of the polynucleotide into a presumptive zygote or one blastomere of a two-cell embryo; or (ii) administration of a gene therapy vector comprising the polynucleotide to a presumptive zygote or two-cell embryo.

[0009] In some such methods, the polynucleotide is introduced via administration of a gene therapy vector. In some such methods, the gene therapy vector comprises: (i) a polynucleotide complex; or (ii) a viral vector. In some such methods, the gene therapy vector is a polynucleotide complex. Optionally, the polynucleotide complex is a lipid nanoparticle comprising the polynucleotide and lipids. In some such methods, the gene therapy vector is a viral vector, and the viral vector is selected from the group consisting of a retrovirus, an adenovirus, a herpes simplex virus, a pox virus, a vaccinia virus, a lentivirus, or an adeno-associated virus (AAV).

[0010] Some such methods further comprise a nuclease agent. Tn some such methods, the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or a nucleic acid encoding the Cas protein; and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such methods, the polynucleotide is genomically integrated. In some such methods, the polynucleotide is extrachromosomal.

[0011] In some such methods, the embryo is a mammalian embryo. Optionally, the mammalian embryo is a bovine embryo. Optionally, the mammalian embryo is a human embryo.

[0012] Also provided herein are compositions for use in the described methods for increasing METTL7A in an embryo to improve in vitro produced embryo competence. Some such compositions comprise METTL7A protein for use in the methods. Some such methods comprise a polynucleotide encoding METTL7A for use in the methods.BRIEF DESCRIPTION OF THE FIGURES

[0013] Fig. 1 shows that exogenous METTL7A improves the developmental potential of bovine IVP embryos. Panel A shows the cleavage of METTL7AOEembryos (n=29) compared to the vehicle control (n=18). Panel B shows the blastocyst rate of METTL7AOEembryos (n=l 1), compared to the control (n=l 1). Panel C shows a representative image of bovine METTL7AOEembryos, scale bar = 100 pm. Panel D shows immunostaining analysis of CDX2 (trophectoderm, TE marker) and SOX2 (inner cell mass, ICM marker) in METTL7AOEembryos compared to the control, scale bar = 50pm. Panel E shows the total TE cell number counts between METTL7AOEblastocysts (n=5) compared to the control (n=5). Panel F shows the total ICM cell number counts between METTL7AOEblastocysts (n=5) compared to the control (n=5). Panel G shows the TE / ICM cell number ratio between METTL7AOEblastocysts compared to the control. Panel H shows a representative bright field image of day 12 (El 2) elongated embryos from METTL7AOEand control embryo transfer, scale bar = 500 pm. Panel I shows the serum INF-tau levels of surrogate cows with METTL7AOEembryo transfer (n=2) compared to the control (n=2) on the day of flushing. Panel J shows immunostaining analysis of CDX2 (TEmarker), S0X2 (embryonic disc marker), and GATA6 (hypoblast marker) in E12 embryos flushed out from the METTL7AOEand control embryo transfer, scale bar = 50pm.

[0014] Fig. 2 shows the transcriptomic analysis of METTL7AOEembryos at 2-, 8-cell, and ICM / TE compared to control. Panel A shows a heatmap of the samples from the same stages of bovine embryos from the METTL7AOEand control group. Panel B shows principal component analysis (PCA) of the transcriptomes of METTL7AOEand control embryos at different developmental stages. Panels C-F show volcano plots showing the number of up- or down- regulated genes in METTL7AOEembryos compared to control at 2-cell (Panel C), 8-cell (Panel D), ICM (Panel E) and TE (Panel F) stages (FDR < 0.05, |log2FC| > 1). The most significant up-regulated genes in METTL7AOEembryos compared to the control are highlighted. Panels G- I show the top GO terms of up- and down-regulated genes METTL7AOEembryos compared to the control at 8-cell (Panel G), ICM (Panel H) and TE (Panel I) stages.

[0015] Fig. 3 shows that exogenous METTL7A reduces mitochondrial stress and decreases superoxide level of bovine pre-implantation embryos. Panel A. Experimental scheme of injecting METTL7A IVT-RNA into one blastomere of 2-cell embryos. Panel B. Immunostaining analysis of F-actin, mitochondrial stress marker (HIFla), and METTL7A-6xHisTag in METTL7AOEblastomeres and control. The red arrow points to condensed chromatin and degradation of actin filament, scale bar = 50pm. In the 12 hours following injection, METTL7AOEblastomeres developed through 1 or 2 cell cycles, while non-injected blastomeres were arrested (n=5, 4 / 5 or 80%) with the described phenotype. Panel C Experimental scheme of zygotic injection and western blot analysis. Panels D and E show Western blot analysis of Succinate Dehydrogenase Complex Flavoprotein Subunit A (SDHA, a marker for mitochondrial respiratory activity) in METTL7AOEblastocysts (n=6) compared to control (n=7). Panel F shows immunostaining analysis of superoxide level measured by MitoSox green in METTL7AOEembryos and control at 8-cell (n = 13 embryos for both groups) and blastocyst stage (n = 10 embryos for both groups). Scale bar, 50pm. Panels G and H show the quantification of superoxide levels in METTL7AOEembryos and control at 8-cell (Panel G) and blastocyst stage (Panel H). Panels I and J show the GSH level in METTL7AOEembryos and control at 8-cell (n=6 for both groups) (Panel I) and blastocyst stage (n=3 for both groups) (Panel J).

[0016] Fig. 4 shows that exogenous METTL7A attenuates DNA damage and promotes cell cycle progression. Panel A shows immunostaining analysis of F-actin, yH2A.X (DNA damage marker) and METTL7A-6xHisTag in METTL7AOEblastomeres (n=5) and control (METTL7AOEnegative blastomeres; non-injected and IVF embryos), scale bar = 50pm. Panels B and C show Western blot analysis and quantification of yH2A.X in METTL7AOEembryos (n=3) and vehicle controls (n=4) at blastocyst stage, 7 days after zygotic injection. Panel D shows immunostaining analysis of p-Chkl (cell cycle checkpoint marker) in M TTL7AOEand IVF control embryos at the cleavage stage, scale bar = 50pm. Panels E and F show immunostaining analysis of the proliferative cells (Ki67+) in METTL7AOEblastocyst and control group. In thel2 hours foilwing zygotic injection, METTL7AOEblastomeres developed through 1 or 2 cell cycles, while non-injected blastomeres were arrested (n = 5).

[0017] Fig. 5 shows the normal expression dynamics of METTL7A throughout bovine embryo development and W\aiMETTL7A knockout (METTL7AKO) reduces embryo competence. Panel A shows the cleavage rate of METTL7AKOembryos compared to the control. Panel B shows the blastocyst rate of METTL7AKOembryos compared to the control. Panel C shows the expression dynamics of METTL7A in bovine oocytes and pre-implantation embryo development.

[0018] Fig. 6 shows relevant control experiments related to the studies. Panel A shows an example of ICM isolated from a blastocyst by blastomere biopsy. Panel B shows confirmation of TE and ICM isolation by immunostaining analysis of SOX2 and CDX2 markers, respectively. Scale bar = 50pm. Panel C shows the size and integrity of in vitro transcribed METTL7A-6xHis mRNA confirmed with Tapestation analysis.DEFINITIONS

[0019] Unless otherwise defined, all terms of art, notations, and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled inthe art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc. 2001. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and / or parameters unless otherwise noted.

[0020] The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any part of a protein or polypeptide having a particular function or structure.

[0021] Proteins are said to have an “N-terminus” and a “C-terminus.” The term “N-terminus” relates to the start of a protein or polypeptide, terminated by an amino acid with a free amine group (-NH2). The term “C-terminus” relates to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).

[0022] The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0023] Nucleic acids are said to have “5' ends” and “3' ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the “5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3' end” if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNAmolecule, discrete elements are referred to as being “upstream” or 5' of the “downstream” or 3' elements.

[0024] An “open reading frame” or “ORF” is a portion of a DNA which contains a sequence of bases that could potentially encode a protein. As an example, an ORF can be located between the start-code sequence (initiation codon) and the stop-codon sequence (termination codon) of a gene. The term “in frame” refers to coding sequences that are part of the same ORF and could be translated continuously, e.g., as a fusion peptide, propeptide, prepropeptide, etc.

[0025] The term “genomically integrated” refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence is integrated into the genome of the cell and is capable of being inherited by progeny thereof. Any method suitable for stable integration of a nucleic acid into the genome of a cell may be used to form a genomically integrated nucleic acid.

[0026] The term “plasmid” or “vector” includes any known vector including a bacterial vector, a viral vector, an episomal plasmid, an integrative plasmid, or a phage vector. The term “vector” refers to a construct which is capable of delivering, and, optionally, expressing, one or more expressible sequences (e.g., protein coding sequence) in a host cell.

[0027] The term “expression vector” or “expression construct” or “expression cassette” refers to a recombinant nucleic acid containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell or organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, as well as other sequences. Eukaryotic cells are generally known to utilize promoters, enhancers, and termination and polyadenylation signals, although some elements may be deleted, and other elements added, without sacrificing the necessary expression.

[0028] The term “targeting vector” refers to a vector that can be introduced by homologous recombination, non-homologous-end-joining-mediated ligation, or any other means of recombination to a target position in the genome of a cell.

[0029] The term “viral vector” refers to a recombinant nucleic acid that includes at least one element of viral origin and includes elements sufficient for or permissive of packaging into a viral vector particle. The vector and / or particle can be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells either ex vivo or in vivo. Numerous forms of viral vectors are known.

[0030] The term “wild type” includes entities having a structure and / or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0031] The term “endogenous” refers to a nucleic acid sequence that occurs naturally within a cell or animal (e.g., a cow or a human). For example, an endogenous METTL7A sequence of an animal refers to a native METTL 7 A sequence that naturally occurs at the METTL 7 A locus in the animal.

[0032] “Exogenous” molecules or sequences include molecules or sequences that are not normally present in a cell in that form. Normal presence includes presence with respect to the particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence, for example, can include a mutated version of a corresponding endogenous sequence within the cell, such as a humanized version of the endogenous sequence, or can include a sequence corresponding to an endogenous sequence within the cell but in a different form (z.e., not within a chromosome). In contrast, endogenous molecules or sequences include molecules or sequences that are normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions.

[0033] The term “heterologous” when used in the context of a nucleic acid or a protein indicates that the nucleic acid or protein comprises at least two portions that do not naturally occur together in the same molecule. A heterologous sequence can be a sequence which is present in a cell, genome, or gene in the genetic context other than where it naturally occurs. For example, the term “heterologous,” when used with reference to portions of a nucleic acid or portions of a protein, indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other (e.g., joined together) in nature. As one example, a “heterologous” region of a nucleic acid vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid vector could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. A heterologous sequence can be a sequence derived from the same gene and / or cell type but introduced into the cell or a similar cell in a different context, such as on an expression vector or in a different chromosomal location or with a different promoter. Likewise, a “heterologous” region of a protein is a segment of amino acids within or attached to anotherpeptide molecule that is not found in association with the other peptide molecule in nature (e.g., a fusion protein, or a protein with a tag). Similarly, a nucleic acid or protein can comprise a heterologous label or a heterologous secretion or localization sequence.

[0034] The term “locus” refers to a specific location of a gene (or significant sequence), DNA sequence, polypeptide-encoding sequence, or position on a chromosome of the genome of an organism. For example, a “METTL7A locus” may refer to the specific location of METTL7A gene, METTL7A DNA sequence, METTL7A-encoding sequence, or METTL7 A position on a chromosome of the genome of an organism that has been identified as to where such a sequence resides. “METTL7A locus” may comprise a regulatory element of METTL7A gene, including, for example, an enhancer, a promoter, 5' and / or 3' untranslated region (UTR), or a combination thereof.

[0035] The term “gene” refers to a DNA sequence in a chromosome that codes for a product (e g., an RNA product and / or a polypeptide product) and includes the coding region interrupted with non-coding introns and sequence located adjacent to the coding region on both the 5' and 3' ends such that the gene corresponds to the full-length mRNA (including the 5' and 3' untranslated sequences). The term “gene” also includes other non-coding sequences including regulatory sequences (e.g., promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequence, and matrix attachment regions. These sequences may be close to the coding region of the gene (e.g., within 10 kb) or at distant sites, and they influence the level or rate of transcription and translation of the gene.

[0036] The term “allele” refers to a variant form of a gene. Some genes have a variety of different forms, which are located at the same position, or genetic locus, on a chromosome. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ.

[0037] A “promoter” is a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other regions which influence the transcription initiation rate. The promoter sequences disclosed herein modulate transcription of an operably linked polynucleotide. A promoter can be active inone or more of the cell types disclosed herein (e.g., a eukaryotic cell, a non-human mammalian cell, a human cell, a rodent cell, a pluripotent cell, a one-cell stage embryo, a differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013 / 176772, herein incorporated by reference in its entirety for all purposes.

[0038] “Operable linkage” or being “operably linked” includes juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence).

[0039] The term “variant” refers to a nucleotide sequence differing from the sequence most prevalent in a population (e.g., by at least one nucleotide) or a protein sequence different from the sequence most prevalent in a population (e.g., by at least one amino acid).

[0040] The term “fragment” when referring to a protein means a protein that is shorter or has fewer amino acids than the full-length protein. The term “fragment” when referring to a nucleic acid means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, an N-terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N- terminal end of the protein), or an internal fragment.

[0041] “Sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0042] “Percentage of sequence identity” includes the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0043] A “homologous” sequence (e.g., nucleic acid sequence) includes a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Homologous sequences can include, for example, orthologous sequence and paralogous sequences. Homologous genes, for example, typically descend from a common ancestral DNA sequence, either through a speciation event (orthologous genes) or a genetic duplication event (paralogous genes). “Orthologous” genes include genes in different species that evolved from a common ancestral gene by speciation. Orthologs typically retain the same function in the course of evolution. “Paralogous”genes include genes related by duplication within a genome. Paralogs can evolve new functions in the course of evolution.

[0044] The terms “substantial identity” and “substantially identical,” as used with reference to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0045] As applied to polypeptides, the terms “substantial identity” and “"substantially identical” mean that two peptide sequences, when optimally aligned, share at least about 90% sequence identity, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.

[0046] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is thecomputer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and 1997 Nucleic Acids Res. 25:3389-3402.

[0047] The term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a non-polar residue. The term “isolated” with respect to proteins and nucleic acid refers to proteins and nucleic acids that are relatively purified with respect to other bacterial, viral, or cellular components that may normally be present in situ, up to and including a substantially pure preparation of the protein and the polynucleotide. The term “isolated” also includes proteins and nucleic acids that have no naturally occurring counterpart, have been chemically synthesized and are thus substantially uncontaminated by other proteins or nucleic acids, or has been separated or purified from most other cellular components with which they are naturally accompanied (e.g., other cellular proteins, polynucleotides, or cellular components).

[0048] The term “zzz vitro" includes artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube). The term “ / > / vzvo” includes natural environments (e.g., organism or body) and to processes or reactions that occur within a natural environment. The term “ex vivo” includes cells that have been removed from the body of an individual and to processes or reactions that occur within such cells.

[0049] The term “reporter gene” refers to a nucleic acid having a sequence encoding a gene product (e.g., an enzyme or a detectable protein; e.g., a fluorescent protein) that is easily and quantifiably assayed when a construct comprising the reporter gene sequence operably linked to a heterologous promoter and / or enhancer element is introduced into cells containing (or whichcan be made to contain) the factors necessary for the activation of the promoter and / or enhancer elements. Examples of reporter genes include, but are not limited, to genes encoding betagalactosidase (lacZ), the bacterial chloramphenicol acetyltransferase (cat) genes, luciferase genes (e.g., nano luciferase), genes encoding beta-glucuronidase (GUS), and genes encoding fluorescent proteins. A “reporter protein” refers to a protein encoded by a reporter gene.

[0050] The term “recombination” includes any process of exchange of genetic information between two polynucleotides and can occur by any mechanism. Recombination in response to double-strand breaks (DSBs) occurs principally through two conserved DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). See Kasparek & Humphrey (2011) Seminars in Cell & Dev. Biol. 22:886-897, herein incorporated by reference in its entirety for all purposes. Likewise, repair of a target nucleic acid mediated by an exogenous donor nucleic acid can include any process of exchange of genetic information between the two polynucleotides.

[0051] Recombination can occur via homology directed repair (HDR) or homologous recombination (HR). HDR or HR includes a form of nucleic acid repair that can require nucleotide sequence homology, uses a “donor” molecule as a template for repair of a “target” molecule (i.e., the one that experienced the double-strand break), and leads to transfer of genetic information from the donor to target. Without wishing to be bound by any particular theory, such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and / or synthesis-dependent strand annealing, in which the donor is used to resynthesize genetic information that will become part of the target, and / or related processes. In some cases, the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA. See Wang et al. (2013) Cell 153:910-918; Mandalos et al. (2012) PLOS ONE 7:e45768:l- 9; and Wang et al. (2013) Nat Biotechnol. 31 :530-532, each of which is herein incorporated by reference in its entirety for all purposes.

[0052] As used herein, a “control” as in a control sample or a control subject (e.g., mouse) is a comparator for a measurement, e.g., a diagnostic measurement of a sign or symptom of a disease (e.g., dystonia). In certain aspects, a control can be a subject sample from the same subject an earlier time point, e.g., before a treatment intervention. In certain aspects, a control can be a measurement from a normal subject, i.e., a subject not having the disease of the treatedsubject, to provide a normal control, e.g., behavioral activity in a subject (e.g., overt dystonia in a mouse). In certain aspects, a control can be an untreated subject with the same disease. In certain aspects, a control can be a subject treated with a different therapy, e.g., the standard of care. In certain aspects, the control is matched for certain factors to the subject being tested, e.g., age, gender. Selection of an appropriate control is within the ability of those of skill in the art.

[0053] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients. The transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified elements recited in the claim and those that do not materially affect the basic and novel character! stic(s) of the claimed invention. Thus, the term “consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”

[0054] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which it does not.

[0055] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.

[0056] In general, the term “about” indicates variation in a quantity of a component of a composition not having a significant effect on the activity or stability of the composition. For example, “about” can mean within 1 standard deviation. Alternatively, “about” can mean a range of up to 0 to 20%, 0 to 10%, 0 to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. When the specification discloses a specific value for a parameter, the specification should be understood as alternatively disclosing the parameter at “about” that value. All ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions, such as “not including the endpoints”; thus, for example, “within 10-15” or “from 10 to 15” includes the values 10 and 15.

[0057] The term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0058] The term “or” refers to any one member of a particular list and also includes any combination of members of that list.

[0059] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof.

[0060] Statistically significant means p <0.05.

[0061] In the event of a conflict between a sequence in the application and an indicated accession number or position in an accession number, the sequence in the application predominates.DETAILED DESCRIPTIONI. Overview

[0062] Provided herein are methods for improving in vitro produced embryo competence by increasing METTL7A expression. Also provided are compositions comprising METTL 7 A for use in such methods.

[0063] IVF is one of the most used assisted reproductive technologies to achieve a successful pregnancy, both in humans and a number of agricultural species. While IVF procedures are considered safe, embryos developing in vitro are exposed to conditions during culture that are not normally experienced in vivo. One of the most well-known deleterious conditions to which IVF embryos are exposed in in vitro culture is oxidative stress. Numerous approaches have been explored in prior attempts to mitigate the increased oxidative stress that exists in vitro, including reducing oxygen tension in incubators, co-culturing with additional protective cell types, and addition of media supplements that can act as ROS scavengers. However, none of these methods have fully overcome the competence issues associated with in vitro produced (IVP) embryos.

[0064] Studies described in the Examples demonstrate, for the first time, that METTL7A modulates expression of genes involved in embryonic cell mitochondrial pathways and promotes trophectoderm development. Surprisingly, it was discovered that METTL7A alleviates mitochondrial stress and DNA damage and promotes cell cycle progression during embryo cleavage. This novel control of mitochondria stress elimination mechanisms, regulated by METTL7A, is particularly advantageous in that it activates a constellation of factors within the embryo to reduce oxidative stress, rather than currently employed methods that add anexogenous ROS scavenger with limited capacity to neutralize oxidative species. Critically, embryos with overexpression of METTL7A, generated by the compositions and methods described herein, produced normal pregnancy through conceptus elongation following embryo transfer to recipients.II. Methyltransferase-like Protein Z4 (METTL7A)

[0065] Methyltransferase-like protein 7A (METTL7A), also known as thiol methyltransferase 1A (TMT1A), is a recently discovered and characterized protein that is involved in lipid droplet formation and possesses S-adenosyl-L-methionine (SAM)-dependent thiol methyltransferase activity. Limited studies to date have suggested METTL7A can promote cell survival and is associated with successful stem cell trajectory reprogramming. However, the complete biological function of METTL7A is largely unknown, and the role of METTL7A during early embryonic development was unexplored prior to the studies disclosed herein.

[0066] Bovine (Bos taurus) METTL7A maps to chromosome 5 (NCBI RefSeq Gene ID 613844; assembly ARS-UCD2.0 (GCF_002263795.3); location NC_037332.1(28996015..29007833, complement). Bovine METTL7A protein has been assigned UniProt accession number Q3MHK8. The canonical amino acid sequence (NP 001030516.1) is set forth in SEQ ID NO: 1. An exemplary mRNA (cDNA) encoding the canonical protein is assigned NCBI Accession No. NM_001035439.1 and is set forth in SEQ ID NO: 2. The canonical, full- length bovine METTL7A protein set forth in SEQ ID NO: 1 has 244 amino acids, including a methyltransferase type 11 domain (amino acids 75-172). Delineations between domains and interacting regions are as designated in UniProt. Reference to bovine METTL7A includes the canonical (wild type) form, as well as all allelic forms and isoforms. Any other forms of bovine METTL7A have amino acids numbered for maximal alignment with the wild type form, aligned amino acids being designated the same number.

[0067] Human METTL 7 A maps to 12ql3.12 on chromosome 12 (NCBI RefSeq Gene ID25840; assembly GRCh38.pl4 (GCF_000001405.40); location NC_000012.12(50925015..50932508). Human METTL7A protein has been assigned UniProt accession number Q9H8H3. The canonical amino acid sequence (NP 054752.3) is set forth in SEQ ID NO: 3. An exemplary mRNA (cDNA) encoding the canonical protein is assigned NCBI Accession No. NM_014033.4 and is set forth in SEQ ID NO: 4. An exemplary coding sequence (CDS) (CCDS8804.1) is set forth in SEQ ID NO: 5. The canonical, full-length human METTL7Aprotein set forth in SEQ ID NO: 3 has 244 amino acids, including a signal peptide (amino acids 1-29) and the mature protein chain (amino acids 30-244). Delineations between domains and interacting regions are as designated in UniProt. Reference to human METTL7A includes the canonical (wild type) form, as well as all allelic forms and isoforms. Any other forms of human METTL7A have amino acids numbered for maximal alignment with the wild type form, aligned amino acids being designated the same number.

[0068] Any ortholog of METTL7A in a species in which in vitro fertilization techniques are applied may be used in the described methods and compositions for that species. Such examples include, but are not limited to, mouse (NCBI RefSeq Gene ID 70152), rat (NCBI RefSeq Gene ID 315306), dog (NCBI RefSeq Gene ID 100685262), horse (NCBI RefSeq Gene ID 100060363), and non-human primates, such as, for example, rhesus monkey (NCBI RefSeq Gene ID 693894).III. Methods for Increasing METTL7A in In Vitro Produced Embryos

[0069] Provided herein are methods for improving in vitro produced embryo competence comprising increasing METTL7A in an embryo. Those of skill in the art will recognize that increasing the amount of a protein in a cell can be accomplished by several methods, including but not limited to, introduction of endogenous protein into the cell, introduction of nucleic acids (e g., DNA or RNA) encoding the protein into the cell in a transient form (e.g., mRNA or a DNA plasmid), or genetic modification of the cell (e g., nuclease-mediated recombination).A. Introduction of Exogenous METTL7A Protein

[0070] In some embodiments, exogenous METTL7A protein is introduced into the embryo. Methods of protein production and purification (see, e.g., Hardin et al., eds. Cloning, Gene Expression, and Protein Purification (2001)) are routine and within the skill of those who would practice the methods described herein, or METTL7A protein can be procured through commercial production services using sequence information provided elsewhere herein.Compositions comprising exogenous METTL7A protein are described elsewhere herein. In some embodiments, introducing METTL7A protein into an embryo comprises microinjection of METTL7A into a presumptive zygote or one blastomere of a two-cell embryo. Those skilled in the art will recognize that any other suitable means of introducing exogenous METTL7A protein into the embryo can be used, so long as the method of introducing does not negatively affectembryo competence when compared to a control embryo. Exogenous METTL7A protein can be introduced alone or in combination with any other embodiments of METTL7A increasing compositions described herein.B. Introduction of Nucleic Acids Encoding METTL7 A

[0071] In some embodiments, a polynucleotide encoding METTL7A is introduced into the embryo. In some embodiments, the polynucleotide encoding METTL7A is DNA. In some embodiments, the polynucleotide encoding METTL7A is RNA. Polynucleotides encoding METTL7A can also include DNA / RNA hybrids or derivatives of DNA and / or RNA. Compositions comprising exogenous METTL7A protein are further described elsewhere herein.

[0072] In some embodiments, introducing the polynucleotide encoding METTL7A comprises microinjection of the polynucleotide into a presumptive zygote or one blastomere of a two-cell embryo. The microinjected polynucleotide can be a naked polynucleotide, a modified polynucleotide, a vector comprising the polynucleotide, or a polynucleotide complex. In some embodiments, introducing the polynucleotide encoding METTL7A comprises administration of a gene therapy vector comprising the polynucleotide to a presumptive zygote or two-cell embryo. Those skilled in the art will recognize that any other suitable means of introducing a nucleic acid encoding METTL7A into the embryo can be used, so long as the method of introducing does not negatively affect embryo competence when compared to a control embryo. Introduction of nucleic acids encoding METTL7A via a gene therapy vector is advantageous in that METTL7A can be simultaneously increased in numerous embryos, as compared to microinjection techniques.

[0073] In some embodiments, the polynucleotide is introduced into the embryo in a gene therapy vector. A vector can comprise additional sequences such as, for example, replication origins, promoters, genes encoding antibiotic resistance, and / or reporter genes.

[0074] Some vectors may be circular. Alternatively, the vector may be linear. The vector can be a naked polynucleotide. The vector can be in the packaged for delivered via a polynucleotide complex, such as, for example a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.

[0075] In one embodiment, the vector is a lipid nanoparticle (LNP) comprising the polynucleotide encoding METTL7A. The lipid nanoparticle can, however, comprise METTL7Ain any form (e.g., protein, DNA, or RNA). The polynucleotide can be modified to comprise one or more stabilizing modifications familiar in the art and described elsewhere herein. Delivery through such methods can result in transient METTL 7 A expression, which may be advantageous in allowing unaltered development in vivo (i.e., a return to normal gene expression postimplantation as directed by cues in utero) following in vitro culture of the embryo.

[0076] Lipid formulations can protect biological molecules from degradation while improving their cellular uptake. Lipid nanoparticles are particles comprising a plurality of lipid molecules physically associated with each other by intermolecular forces. These include microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations which contain cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the length of time for which nanoparticles can exist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in WO 2016 / 010840 Al and WO 2017 / 173054 Al, each of which is herein incorporated by reference in its entirety for all purposes. An exemplary lipid nanoparticle can comprise a cationic lipid and one or more other components. In one example, the other component can comprise a helper lipid such as cholesterol. In another example, the other components can comprise a helper lipid such as cholesterol and a neutral lipid such as DSPC. In another example, the other components can comprise a helper lipid such as cholesterol, an optional neutral lipid such as DSPC, and a stealth lipid such as SO 10, S024, S027, S031, or S033. In some embodiments, the LNP may contain one or more or all of the following: (i) a lipid for encapsulation and for endosomal escape; (ii) a neutral lipid for stabilization; (iii) a helper lipid for stabilization; and (iv) a stealth lipid. See, e.g., Finn et al. (2018) Cell Rep. 22(9y.2221 -2235 , herein incorporated by reference in its entirety for all purposes.

[0077] In some embodiments, the vector is a viral vector. The vectors can be, for example, viral vectors such as adeno-associated virus (AAV) vectors. The AAV may be any suitable serotype and may be a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV). Other exemplary viruses / viral vectors include retroviruses, lentiviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The viruses can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells (i.e., gametes or zygotes can be infected to increase METTL7A). The viruses can integrate into the host genome or alternatively do not integrate into the host genome. Such viruses can also be engineered to have reduced immunity. The viruses can be replication-competent or can be replication-defective (e.g., defective in one or more genes necessary for additional rounds of virion replication and / or packaging). Viruses can cause transient expression, long-lasting expression (e.g., at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or permanent expression. A viral vector may be genetically modified from their wild type counterparts. For example, the viral vector may comprise an insertion, deletion, or substitution of one or more nucleotides to facilitate cloning or such that one or more properties of the vector is changed. Such properties may include packaging capacity, transduction efficiency, immunogenicity, genome integration, replication, transcription, and translation. In some embodiments, a portion of the viral genome may be deleted such that the virus is capable of packaging exogenous sequences having a larger size. In some embodiments, the viral vector may have an enhanced transduction efficiency. In some embodiments, viral genes (such as integrase) that promote integration of the viral sequence into a host genome may be mutated such that the virus becomes non-integrating. In some embodiments, the viral vector may be replication defective. In some embodiments, the viral vector may comprise exogenous transcriptional or translational control sequences to drive expression of coding sequences in the vector. In some examples, the virus may be helper-dependent. For example, the virus may need one or more helper viruses to supply viral components (such as viral proteins) required to amplify and package the vectors into viral particles. In other examples, the virus may be helper-free. For example, the virus may be capable of amplifying and packaging the vectors without a helper virus. In some examples, the vector system described herein may also encode the viral components required for virus amplification and packaging.

[0078] Adeno-associated viruses (AAVs) are endemic in multiple species including human and non-human primates (NHPs). At least 12 natural serotypes and hundreds of natural variants have been isolated and characterized to date. See, e.g., Li et al. (2020) Nat. Rev. Genet. 21:255- 272, herein incorporated by reference in its entirety for all purposes. AAV particles are naturally composed of a non-enveloped icosahedral protein capsid containing a single-stranded DNA (ssDNA) genome. The DNA genome is flanked by two inverted terminal repeats (ITRs) which serve as the viral origins of replication and packaging signals. The rep gene encodes fourproteins required for viral replication and packaging whilst the cap gene encodes the three structural capsid subunits which dictate the AAV serotype, and the Assembly Activating Protein (AAP) which promotes virion assembly in some serotypes.

[0079] Recombinant AAV (rAAV) is currently one of the most commonly used viral vectors to deliver transgenes to target cells. Indeed, rAAV vectors are composed of icosahedral capsids similar to natural AAVs, but rAAV virions do not encapsidate AAV protein-coding or AAV replicating sequences. These viral vectors are non-replicating. The only viral sequences required in rAAV vectors are the two ITRs, which are needed to guide genome replication and packaging during manufacturing of the rAAV vector. rAAV genomes are devoid of AAV rep and cap genes, rendering them non-replicating in vivo. rAAV vectors are produced by expressing rep and cap genes along with additional viral helper proteins in trans, in combination with the intended transgene cassette flanked by AAV ITRs.

[0080] In rAAV genomes for transgene delivery (e.g., Cre), a gene expression cassette is placed between ITR sequences. Typically, rAAV genome cassettes comprise of a promoter to drive expression of a transgene, followed by polyadenylation sequence. The ITRs flanking a rAAV expression cassette are usually derived from AAV2, the first serotype to be isolated and converted into a recombinant viral vector. Since then, most rAAV production methods rely on AAV2 / A / i-based packaging systems. See, e.g., Colella et al. (2017) Mol. Then Methods Clin. Dev. 8:87-104, herein incorporated by reference in its entirety for all purposes.

[0081] The specific serotype of a recombinant AAV vector influences its in vivo tropism to specific tissues. AAV capsid proteins are responsible for mediating attachment and entry into target cells, followed by endosomal escape and trafficking to the nucleus. Thus, the choice of serotype when developing a rAAV vector will influence what cell types and tissues the vector is most likely to bind to and transduce when injected in vivo.

[0082] Multiple serotypes of AAV have been identified. These serotypes differ in the types of cells they infect (i.e., their tropism), allowing preferential transduction of specific cell types. The term AAV includes, for example, AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64Rl, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2 / 8, AAVrhlO, AAVLK03, AV10, AAV11, AAV12, rhlO, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats(TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. An “AAV vector” as used herein refers to an AAV vector comprising a heterologous sequence not of AAV origin ( / .(?., a nucleic acid sequence heterologous to AAV), typically comprising a sequence encoding a heterologous polypeptide (e.g., METTL7A). The AAV vector may comprise an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64Rl, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2 / 8, AAVrhlO, AAVLK03, AV10, AAV11, AAV12, rhlO, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV capsid sequence. In general, the heterologous nucleic acid sequence (the transgene) is flanked by at least one, and generally by two, AAV inverted terminal repeat sequences (ITRs). An AAV vector may either be single-stranded (ssAAV) or self-complementary (scAAV).

[0083] Tropism can be further refined through pseudotyping, which is the mixing of a capsid and a genome from different viral serotypes. For example, AAV2 / 5 indicates a virus containing the genome of serotype 2 packaged in the capsid from serotype 5. Use of pseudotyped viruses can improve transduction efficiency, as well as alter tropism. Hybrid capsids derived from different serotypes can also be used to alter viral tropism. For example, AAV-DJ contains a hybrid capsid from eight serotypes and displays high infectivity across a broad range of cell types in vivo. AAV-DJ8 is another example that displays the properties of AAV-DJ but with enhanced brain uptake. AAV serotypes can also be modified through mutations. Examples of mutational modifications of AAV2 include Y444F, Y500F, Y730F, and S662V Examples of mutational modifications of AAV3 include Y705F, Y731F, and T492V. Examples of mutational modifications of AAV6 include S663V and T492V Other pseudotyped / modified AAV variants include AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, AAV8.2, and AAV / SASTG.Such modification of a rAAV vector to alter tropism and / or transduction efficiency ( / .<?., based on the species of gamete or zygote to be infected) will be well-understood and routine for a skilled person practicing the claimed invention.

[0084] Once in the nucleus, the ssDNA genome is released from the virion and a complementary DNA strand is synthesized to generate a double-stranded DNA (dsDNA) molecule. Double-stranded AAV genomes naturally circularize via their ITRs and become episomes which will persist extrachromosomally in the nucleus. Therefore, for episomal gene expression, rAAV-delivered rAAV episomes provide long-term, promoter-driven gene expressionin non-dividing cells. However, this rAAV-delivered episomal DNA is diluted out as cells divide. Accordingly, in some embodiments, the polynucleotide encoding METTL7Ais extrachromosomal following introduction.

[0085] In some embodiments, the methods further comprise introducing a nuclease agent. Any suitable nuclease agent can be used in the described methods and are described elsewhere herein. In some embodiments, the nuclease agent is used to facilitate genomic integration of the polynucleotide encoding METTL7A, such that the polynucleotide is genomically integrated following introduction. In some embodiments, the nuclease agent is used to increase expression of METTL7A through alternative means, such as, for example, modification or replacement of enhancer or promoter elements.

[0086] In some embodiments, the nuclease agent comprises a zinc finger nuclease (ZFN). In some embodiments, the nuclease agent comprises a transcription activator-like effector nuclease (TALEN). In some embodiments, the nuclease agent comprises a Cas protein or a nucleic acid encoding the Cas protein and a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0087] The introduction of METTL7A, by any of the methods described above, can be performed at various times during the IVF process (e.g., in gametes, zygotes, or embryos). In some embodiments, METTL7A is introduced into a gamete (e.g., oocytes or spermatozoa). METTL7A can be introduced into oocytes at any stage of maturation (e.g., germinal vesicle (GV), metaphase I (MI), or metaphase II (Mil)). In some embodiments, METTL7A is introduced into a zygote (i.e., a one-cell stage embryo). In some embodiments, METTL7A is introduced into a two-cell stage embryo. In some embodiments, METTL7A is introduced into an eight-cell stage embryo. In some embodiments, METTL7A is introduced into a morula stage embryo. In some embodiments, METTL7A is introduced into a blastula stage embryo.

[0088] A gamete, zygote, or embryo provided for the methods described above can be from any type of animal or mammal. Methods for harvest of gametes for in vitro fertilization and maturation will be familiar to those of skill in the art. Mammals include, for example, humans, non-human mammals, non-human primates, monkeys, apes, cats, dogs, horses, bulls, deer, bison, sheep, rabbits, rodents (e.g., but not limited to, mice, rats, hamsters, and guinea pigs), andlivestock (e.g., but not limited to, bovine species such as cows and steer; ovine species such as sheep and goats; and porcine species such as pigs and boars). Birds include, for example, chickens, turkeys, ostrich, geese, and ducks. Domesticated animals and agricultural animals are also included. The term “non-human mammal” excludes humans. In some embodiments, the embryo is a mammalian embryo. In some embodiments, the embryo is from an agricultural animal. In some embodiments, the mammalian embryo is a bovine embryo. In some embodiments, the mammalian embryo is an equine embryo. In some embodiments, the mammalian embryo is an ovine embryo. In some embodiments, the mammalian embryo is a porcine embryo. In some embodiments, the mammalian embryo is a human embryo.IV. Compositions Comprising METTL7 A

[0089] Also provided herein are compositions comprising METTL7A, or nucleic acids encoding METTL7A, for use in the methods described above. Further provided are nuclease agents that can be introduced using the methods described above in combination with nucleic acids encoding METTL7Ato facilitate genomic integration. Alternatively, nuclease agents can be introduced using the methods described above to indirectly increase METTL7A through modification or replacement of, for example, promoter, enhancer, or other non-coding elements that regulate METTL7A expression.

[0090] Any of the compositions described below can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or auxiliaries (e.g., water, IVF media, various buffers, etc.). The formulation can depend on several factors, including but not limited to, the composition (e.g., nucleic acid, protein, LNP, viral vector, etc.) and its related physical properties (e.g., solubility), the cell type into which it is being introduced (e g., oocytes, spermatozoa, zygotes, embryos), the method of introduction, and others. The term “pharmaceutically acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the intended target cell or organism (e.g., embryo).

[0091] The frequency of administration and the number of times compositions comprising METTL7A can depend on several factors, such as whether the composition is genomically integrated or transiently present (e.g., nuclease-mediated integration oiMETTL7A coding sequence into a zygote as compared to microinjection of METTL7A protein into a zygote). The introduction of such compositions can be performed one time or multiple times over a period oftime. Intervals may be regular or irregular based on response in the embryo, requirements for dosing of the composition, or other factors.A. METTL7A Protein

[0092] In some embodiments, compositions comprising METTL7A protein are introduced into a gamete, zygote, or embryo as described above. Methods of protein production and purification (see, e.g., Hardin et al., eds. Cloning, Gene Expression, and Protein Purification (2001)) are routine and within the skill of those who would practice the methods described herein, and / or METTL7A protein can be procured through commercial production services using sequence information provided herein. In some such compositions, METTL7A protein comprises the sequence set forth in SEQ ID NO: 1. In some such compositions, METTL7A protein consists of the sequence set forth in SEQ ID NO: 1. In some such compositions, the METTL7A protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some such compositions, METTL7A protein comprises the sequence set forth in SEQ ID NO: 3. In some such compositions, METTL7A protein consists of the sequence set forth in SEQ ID NO: 3. In some such compositions, the METTL7A protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3.

[0093] Any protein coding sequence for METTL7A for a species of interest can be found in an online repository, such as UniProt, and subsequently synthesized to create a composition for use in the methods above. The species from which the METTL7A protein is derived and the gamete, zygote, or embryo into which it will be introduced need not match, particularly if a METTL7A from a particular species has especially desirable properties (e.g., enhanced stability in vitro, longer half-life in the cell or cells, etc.). Additionally, any biologically active fragment of METTL7A that improves embryo competence can be used in place of full-length METTL7A. Likewise, the nucleic acids described below may encode full-length METTL7A or any biologically active fragment thereof that is effective in improving embryo competence.B. Nucleic Acids Encoding METTL7A

[0094] In some embodiments, compositions comprising nucleic acids encoding METTL7A are introduced into a gamete, zygote, or embryo as described above. A nucleic acid encodingMETTL7A comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Such nucleic acids can be DNA, RNA, or hybrids or derivatives of either DNA or RNA. Optionally, in some embodiments, the nucleic acid can be codon-optimized for efficient translation into protein in a particular cell or organism. As a non-limiting example, the nucleic acid can be modified to substitute codons having a higher frequency of usage in a cell (e.g., an embryonic cell or embryo) of a particular organism, such as, for example, a mammalian cell, a bovine cell, a human cell, a rodent cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. Any portion or fragment of a nucleic acid molecule can be produced by: (1) isolating the molecule from its natural milieu; (2) using recombinant DNA technology (e.g., but not limited to, PCR amplification or cloning); or (3) using chemical synthesis methods. Non-limiting exemplary methods of in vitro synthesis are provided in the Examples herein. Nucleic acids can comprise modifications for improved stability or reduced immunogenicity. Non-limiting examples of modifications include: (1) alteration or replacement of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage; (2) alteration or replacement of a constituent of a ribose sugar such as alteration or replacement of the 2’ hydroxyl on the ribose sugar; (3) replacement of the phosphate moiety with dephospho linkers; (4) modification or replacement of a naturally occurring nucleobase; (5) replacement or modification of a ribose-phosphate backbone; (6) modification of the 3’ end or 5’ end of the oligonucleotide (e.g., but not limited to, removal, modification or replacement of a terminal phosphate group or conjugation of a moiety); and (7) modification of the sugar. Those of skill in the art will be familiar with the introduction of such nucleic acid modifications.

[0095] In some such compositions, the nucleic acid encoding METTL7A comprises the sequence set forth in SEQ ID NO: 2. In some such compositions, the nucleic acid encoding METTL7A consists of the sequence set forth in SEQ ID NO: 2. In some such compositions, the nucleic acid encoding METTL7A is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In some such compositions, nucleic acids encoding METTL7A comprises the sequence set forth in SEQ ID NO: 4 or 5. In some such compositions, the nucleic acid encoding METTL7A consists of the sequence set forth in SEQ ID NO: 4 or 5. In some such compositions, the nucleic acid encoding METTL7A is at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4 or 5. Any nucleic acid encoding METTL7A for a species of interest can be found in an online repository, such as the National Center for Biotechnology (NCBI) Gene database, and subsequently synthesized to create a composition for use in the methods above. The species from which the METTL7A coding sequence is derived and the gamete, zygote, or embryo into which it will be introduced need not match, particularly if a METTL7Afrom a particular species has especially desirable properties (e.g., enhanced stability in vitro, longer half-life in the cell or cells, etc.). Additionally, any nucleic acid encoding a biologically active fragment of METTL7A that improves embryo competence can be used in place of a nucleic acid encoding full-length METTL7A.

[0096] In some embodiments, the nucleic acids can be in the form of an expression construct as defined elsewhere herein. As a non-limiting example, the nucleic acids can include regulatory regions that control expression of the nucleic acid molecule (e g., but not limited to, transcription or translation control regions, 5' and / or 3' UTRs, etc.), full-length or partial coding regions, and combinations thereof. As a non-limiting example, the nucleic acids can be operably linked to a promoter active in a cell or organism of interest. Promoters that can be used in such expression constructs include promoters active, for example, in one or more eukaryotic cells, such as a mammalian cell (e.g., a non-human mammalian cell or a human cell), including one or more cells present at any stage (e.g., zygote, two-cell, eight-cell, morula, blastula, etc.) of early embryonic development. Such promoters can be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters.C. Nuclease Agents

[0097] In some embodiments, compositions comprising nuclease agents or nucleic acids encoding nuclease agents are used in the methods described above to genetically modify the genome of the gamete, zygote, or embryo to increase METTL7A.

[0098] Various methods can be used to generate a targeted genetic modification in a gamete, zygote, or embryo genome of interest. Methods of obtaining the targeted genetic modifications can comprise unaided homologous recombination, recombinase-based insertion, and DNA repair-based insertion and are known in the art (Dong et al., 2021, PNAS. 118(22) e2004834117). Recombinase-based insertion can comprise systems and constructs involving site-specific recombinases, including but not limited to, Cre.loxP systems, F p FRT systems, Dre.rox systems,VCreVoxU systems, Gin.gix systems, Bxbl .attP / attB systems, phiCB I , -attP / attB systems. DNA repair-based insertion methods rely upon the activity of a nuclease agent, including but not limited to a Transcription Activator-Like Effector Nuclease (TALEN; see, WO 2010 / 079430; Morbitzer et al. (2010) PNAS 10.1073 / pnas.1013133107; Scholze & Boch (2010) Virulence 1 :428-432; Christian et al. Genetics (2010) 186:757-761; Li etal. (2Q\Q) Nuc. Acids Res. (2010) doi: 10.1093 / nar / gkq704; and Miller et al. (2011) Nature Biotechnology 29 : 143— 148; all of which are herein incorporated by reference), a zinc-finger nuclease (ZFN; see, US20060246567;US20080182332; US20020081614; US20030021776; WO / 2002 / 057308A2; US20130123484; US20100291048; WO / 2011 / 017293 A2; and Gaj etal. (2013) Trends in Biotechnology, 31(7):397-405 each of which is herein incorporated by reference), a meganuclease (see, Guhan and Muniyappa (2003) Grit Rev Biochem Mol Biol 38: 199-248; Lucas et al., (2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard, (1999) Cell Mol Life Sci 55: 1304-26; Stoddard, (2006) 0 Rev Biophys 38:49-95; and Moure et al., (2002) Nat Struct Biol 9:764 each of which is herein incorporated by reference), or a CRISPR / Cas system (see WO 2013 / 176772, WO 2014 / 065596, WO 2014 / 131833, WO 2016 / 106121, WO 2019 / 067910, WO 2020 / 082042, US 2020 / 0270617, WO 2020 / 082041, US 2020 / 0268906, WO 2020 / 082046, US 2020 / 0289628, Cebrian-Serrano and Davies (2017) Mamm. Genome 28(7):247-261, Zetsche et al. (2015) Cell 163(3):759-771, Liu et al. (2019) Nature 566(7743):218-223, and Pausch et al. (2020) Science 369(6501):333- 337 each of which is herein incorporated by reference). Any suitable nuclease agent can be used in the described methods. In some embodiments, the nuclease agent is a CRISPR / Cas system.CRISI’R Cas Systems

[0099] CRISPR / Cas systems include transcripts and other elements involved in the expression of, or directing the activity of, Cas genes. A CRISPR / Cas system can be, for example, a type I, a type II, a type III system, or a type V system (e.g., subtype V-A or subtype V-B). The methods and compositions disclosed herein can employ CRISPR / Cas systems by utilizing CRISPR complexes (comprising a guide RNA (gRNA) complexed with a Cas protein) for site- directed binding or cleavage of nucleic acids. A CRISPR / Cas system targeting an METTL7A gene or locus comprises a Cas protein (or a nucleic acid encoding the Cas protein) and one or more guide RNAs (or DNAs encoding the one or more guide RNAs), with each of the one or more guide RNAs targeting a different guide RNA target sequence in the target genomic locus (e.g., METTL7A gene or locus).

[0100] CRISPR / Cas systems used in the compositions and methods disclosed herein can be non-naturally occurring. A non-naturally occurring system includes anything indicating the involvement of the hand of man, such as one or more components of the system being altered or mutated from their naturally occurring state, being at least substantially free from at least one other component with which they are naturally associated in nature, or being associated with at least one other component with which they are not naturally associated. For example, some CRISPR / Cas systems employ non-naturally occurring CRISPR complexes comprising a gRNA and a Cas protein that do not naturally occur together, employ a Cas protein that does not occur naturally, or employ a gRNA that does not occur naturally.Cas Proteins

[0101] Cas proteins generally comprise at least one RNA recognition or binding domain that can interact with guide RNAs. Cas proteins can also comprise nuclease domains (e.g., DNase domains or RNase domains), DNA-binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Some such domains (e.g., DNase domains) can be from a native Cas protein. Other such domains can be added to make a modified Cas protein. A nuclease domain possesses catalytic activity for nucleic acid cleavage, which includes the breakage of the covalent bonds of a nucleic acid molecule. Cleavage can produce blunt ends or staggered ends, and it can be single-stranded or double-stranded. For example, a wild type Cas9 protein will typically create a blunt cleavage product. Alternatively, a wild type Cpfl protein (e.g., FnCpfl) can result in a cleavage product with a 5-nucleotide 5’ overhang, with the cleavage occurring after the 18th base pair from the PAM sequence on the non-targeted strand and after the 23rd base on the targeted strand. A Cas protein can have full cleavage activity to create a double-strand break at a target genomic locus (e.g., a double-strand break with blunt ends), or it can be a nickase that creates a single-strand break at a target genomic locus.

[0102] Examples of Cas proteins include Cast, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.

[0103] An exemplary Cas protein is a Cas9 protein or a protein derived from a Cas9 protein. Cas9 proteins are from a type II CRISPR / Cas system and typically share four key motifs with a conserved architecture. Motifs 1, 2, and 4 are RuvC-like motifs, and motif 3 is an HNH motif. Exemplary Cas9 proteins are from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AU cyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicehdosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus hal ophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Neisseria meningitidis, or Campylobacter jejuni. Additional examples of the Cas9 family members are described in WO 2014 / 131833, herein incorporated by reference in its entirety for all purposes. Cas9 from S. pyogenes (SpCas9) (e.g., assigned UniProt accession number Q99ZW2) is an exemplary Cas9 protein.

[0104] Cas proteins can be wild type proteins (i.e., those that occur in nature), modified Cas proteins (i.e., Cas protein variants), or fragments of wild type or modified Cas proteins. Cas proteins can also be active variants or fragments with respect to catalytic activity of wild type or modified Cas proteins. Active variants or fragments with respect to catalytic activity can comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the wild type or modified Cas protein or a portion thereof, wherein the active variants retain the ability to cut at a desired cleavage site and hence retain nick-inducing or double-strand-break-inducing activity. Assays for nick-inducing or double-strand-break-inducingactivity are known and generally measure the overall activity and specificity of the Cas protein on DNA substrates containing the cleavage site.

[0105] One example of a modified Cas protein is the modified SpCas9-HFl protein, which is a high-fidelity variant of Streptococcus pyogenes Cas9 harboring alterations (N497A / R661A / Q695A / Q926A) designed to reduce non-specific DNA contacts. See, e.g., Kleinstiver et al. (2016) Nature 529(7587):490-495, herein incorporated by reference in its entirety for all purposes. Another example of a modified Cas protein is the modified eSpCas9 variant (K848A / K1003A / R1060A) designed to reduce off-target effects. See, e.g., Slaymaker et al. (2016) Science 351(6268):84-88, herein incorporated by reference in its entirety for all purposes. Other SpCas9 variants include K855A and K810A / K1003A / R1060A. These and other modified Cas proteins are reviewed, e.g., in Cebrian-Serrano and Davies (2017) Mamm. Genome 28(7):247-261, herein incorporated by reference in its entirety for all purposes. Another example of a modified Cas9 protein is xCas9, which is a SpCas9 variant that can recognize an expanded range of PAM sequences. See, e.g., Hu et al. (2018) Nature 556:57-63, herein incorporated by reference in its entirety for all purposes.

[0106] Cas proteins can be modified to increase or decrease one or more of nucleic acid binding affinity, nucleic acid binding specificity, and enzymatic activity. Cas proteins can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or a Cas protein can be truncated to remove domains that are not essential for the function of the protein or to optimize (e.g., enhance or reduce) the activity of or a property of the Cas protein.

[0107] Cas proteins can comprise at least one nuclease domain, such as a DNase domain. For example, a wild type Cpfl protein generally comprises a RuvC-like domain that cleaves both strands of target DNA, perhaps in a dimeric configuration. Likewise, CasX and Cas generally comprise a single RuvC-like domain that cleaves both strands of a target DNA. Cas proteins can also comprise at least two nuclease domains, such as DNase domains. For example, a wild type Cas9 protein generally comprises a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains can each cut a different strand of double- stranded DNA to make a double-stranded break in the DNA. See, e.g., Jinek et al. (2012) Science 337(6096):816- 821, herein incorporated by reference in its entirety for all purposes.

[0108] One or more of the nuclease domains can be deleted or mutated so that they are no longer functional or have reduced nuclease activity. For example, if one of the nuclease domains is deleted or mutated in a Cas9 protein, the resulting Cas9 protein can be referred to as a nickase and can generate a single-strand break within a double-stranded target DNA but not a doublestrand break (i.e., it can cleave the complementary strand or the non-complementary strand, but not both). If none of the nuclease domains is deleted or mutated in a Cas9 protein, the Cas9 protein will retain double-strand-break-inducing activity. An example of a mutation that converts Cas9 into a nickase is a D10A (aspartate to alanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 from S. pyogenes. Likewise, H939A (histidine to alanine at amino acid position 839), H840A (histidine to alanine at amino acid position 840), or N863 A (asparagine to alanine at amino acid position N863) in the HNH domain of Cas9 from S. pyogenes can convert the Cas9 into a nickase. Other examples of mutations that convert Cas9 into a nickase include the corresponding mutations to Cas9 from S. thermophilus. See, e.g., Sapranauskas et al. (2011) Nucleic Acids Res. 39(21):9275-9282 and WO 2013 / 141680, each of which is herein incorporated by reference in its entirety for all purposes. Such mutations can be generated using methods such as site-directed mutagenesis, PCR-mediated mutagenesis, or total gene synthesis. Examples of other mutations creating nickases can be found, for example, in WO 2013 / 176772 and WO 2013 / 142578, each of which is herein incorporated by reference in its entirety for all purposes.

[0109] Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, a Cas protein can be fused to a cleavage domain. See WO 2014 / 089290, herein incorporated by reference in its entirety for all purposes. Cas proteins can also be fused to a heterologous polypeptide providing increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the Cas protein.

[0110] As one example, a Cas protein can be fused to one or more heterologous polypeptides that provide for subcellular localization. Such heterologous polypeptides can include, for example, one or more nuclear localization signals (NLS) such as the monopartite SV40 NLS and / or a bipartite alpha-importin NLS for targeting to the nucleus, a mitochondrial localization signal for targeting to the mitochondria, an ER retention signal, and the like. See, e.g., Lange et al. (2007) J. Biol. Chem. 282(8):5101-5105, herein incorporated by reference in its entirety forall purposes. Such subcellular localization signals can be located at the N-terminus, the C- terminus, or anywhere within the Cas protein. An NLS can comprise a stretch of basic amino acids, and can be a monopartite sequence or a bipartite sequence. Optionally, a Cas protein can comprise two or more NLSs, including an NLS (e.g., an alpha-importin NLS or a monopartite NLS) at the N-terminus and an NLS (e.g., an SV40 NLS or a bipartite NLS) at the C-terminus. A Cas protein can also comprise two or more NLSs at the N-terminus and / or two or more NLSs at the C-terminus.

[0111] Cas proteins can also be operably linked to a cell-penetrating domain or protein transduction domain. For example, the cell-penetrating domain can be derived from the HIV-1 TAT protein, the TLM cell-penetrating motif from human hepatitis B virus, MPG, Pep-1, VP22, a cell penetrating peptide from Herpes simplex virus, or a polyarginine peptide sequence. See, e.g., WO 2014 / 089290 and WO 2013 / 176772, each of which is herein incorporated by reference in its entirety for all purposes. The cell-penetrating domain can be located at the N-terminus, the C- terminus, or anywhere within the Cas protein.

[0112] Cas proteins can also be operably linked to a heterologous polypeptide for ease of tracking or purification, such as a fluorescent protein, a purification tag, or an epitope tag. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFPl, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent proteins (e.g., mOrange, mKO, Kusabira- Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), and any other suitable fluorescent protein. Examples of tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, SI, T7, V5, VSV-G, histidine (His), biotin carboxyl carrier protein (BCCP), and calmodulin.

[0113] Cas proteins can be provided in any form. For example, a Cas protein can be provided in the form of a protein, such as a Cas protein complexed with a gRNA. Alternatively, a Cas protein can be provided in the form of a nucleic acid encoding the Cas protein, such as an RNA (e.g., messenger RNA (mRNA)) or DNA. Optionally, the nucleic acid encoding the Cas protein can be codon optimized for efficient translation into protein in a particular cell or organism. For example, the nucleic acid encoding the Cas protein can be modified to substitute codons having a higher frequency of usage in a bacterial cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the Cas protein is introduced into the cell, the Cas protein can be transiently, conditionally, or constitutively expressed in the cell.Guide RNAs

[0114] A “guide RNA” or “gRNA” is an RNA molecule that binds to a Cas protein (e.g., Cas9 protein) and targets the Cas protein to a specific location within a target DNA. Guide RNAs can comprise two segments: a “DNA-targeting segment” (also called “guide sequence”) and a “protein-binding segment.” “Segment” includes a section or region of a molecule, such as a contiguous stretch of nucleotides in an RNA. Some gRNAs, such as those for Cas9, can comprise two separate RNA molecules: an “activator-RNA” (e.g., tracrRNA) and a “targeter- RNA” (e.g., CRISPR RNA or crRNA). Other gRNAs are a single RNA molecule (single RNA polynucleotide), which can also be called a “single-molecule gRNA,” a “single-guide RNA,” or an “sgRNA.” See, e.g., WO 2013 / 176772, WO 2014 / 065596, WO 2014 / 089290, WO 2014 / 093622, WO 2014 / 099750, WO 2013 / 142578, and WO 2014 / 131833, each of which is herein incorporated by reference in its entirety for all purposes. A guide RNA can refer to either a CRISPR RNA (crRNA) or the combination of a crRNA and a trans-activating CRISPR RNA (tracrRNA). The crRNA and tracrRNA can be associated as a single RNA molecule (single guide RNA or sgRNA) or in two separate RNA molecules (dual guide RNA or dgRNA). For Cas9, for example, a single-guide RNA can comprise a crRNA fused to a tracrRNA (e.g., via a linker). For Cpfl and CasO, for example, only a crRNA is needed to achieve binding to a target sequence. The terms “guide RNA” and “gRNA” include both double-molecule (i.e., modular) gRNAs and single-molecule gRNAs.

[0115] In systems in which both a crRNA and a tracrRNA are needed, the crRNA and the corresponding tracrRNA hybridize to form a gRNA. In systems in which only a crRNA is needed, the crRNA can be the gRNA. The crRNA additionally provides the single-stranded DNA-targeting segment that hybridizes to the complementary strand of a target DNA. If used for modification within a cell, the exact sequence of a given crRNA or tracrRNA molecule can be designed to be specific to the species in which the RNA molecules will be used. See, e.g., Mali et al. (2013) Science 339(6121):823-826; Jinek et al. (2012) Science 337(6096): 816-821; Hwang et al. (2013) Nat. Biotechnol. 31(3):227-229; Jiang et al. (2013) Nat. Biotechnol. 31(3):233-239; and Cong et al. (2013) Science 339(6121 ):819-823, each of which is herein incorporated by reference in its entirety for all purposes.

[0116] The DNA-targeting segment (crRNA) of a given gRNA comprises a nucleotide sequence that is complementary to a sequence on the complementary strand of the target DNA, as described in more detail below. The DNA-targeting segment of a gRNA interacts with the target DNA in a sequence-specific manner via hybridization (i.e., base pairing). As such, the nucleotide sequence of the DNA-targeting segment may vary and determines the location within the target DNA with which the gRNA and the target DNA will interact. The DNA-targeting segment of a subject gRNA can be modified to hybridize to any desired sequence within a target DNA. Naturally occurring crRNAs differ depending on the CRISPR / Cas system and organism but often contain a targeting segment of between 21 to 72 nucleotides length, flanked by two direct repeats (DR) of a length of between 21 to 46 nucleotides (see, e.g., WO 2014 / 131833, herein incorporated by reference in its entirety for all purposes). In the case of S. pyogenes, the DRs are 36 nucleotides long and the targeting segment is 30 nucleotides long. The 3’ located DR is complementary to and hybridizes with the corresponding tracrRNA, which in turn binds to the Cas protein.

[0117] The DNA-targeting segment can have, for example, a length of at least about 12, at least about 15, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40 nucleotides. Such DNA- targeting segments can have, for example, a length from about 12 to about 100, from about 12 to about 80, from about 12 to about 50, from about 12 to about 40, from about 12 to about 30, from about 12 to about 25, or from about 12 to about 20 nucleotides. For example, the DNA targeting segment can be from about 15 to about 25 nucleotides (e.g., from about 17 to about 20nucleotides, or about 17, 18, 19, or 20 nucleotides). See, e.g., US 2016 / 0024523, herein incorporated by reference in its entirety for all purposes. For Cas9 from S. pyogenes, a typical DNA-targeting segment is between 16 and 20 nucleotides in length or between 17 and 20 nucleotides in length. For Cas9 from S. aureus, a typical DNA-targeting segment is between 21 and 23 nucleotides in length. For Cpfl, a typical DNA-targeting segment is at least 16 nucleotides in length or at least 18 nucleotides in length.

[0118] In one example, the DNA-targeting segment can be about 20 nucleotides in length. However, shorter and longer sequences can also be used for the targeting segment (e.g., 15-25 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). The degree of identity between the DNA-targeting segment and the corresponding guide RNA target sequence (or degree of complementarity between the DNA-targeting segment and the other strand of the guide RNA target sequence) can be, for example, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. The DNA-targeting segment and the corresponding guide RNA target sequence can contain one or more mismatches. For example, the DNA-targeting segment of the guide RNA and the corresponding guide RNA target sequence can contain 1-4, 1-3, 1-2, 1, 2, 3, or 4 mismatches (e.g., where the total length of the guide RNA target sequence is at least 17, at least 18, at least 19, or at least 20 or more nucleotides). For example, the DNA-targeting segment of the guide RNA and the corresponding guide RNA target sequence can contain 1-4, 1-3, 1-2, 1, 2, 3, or 4 mismatches where the total length of the guide RNA target sequence 20 nucleotides.

[0119] TracrRNAs can be in any form (e.g., full-length tracrRNAs or active partial tracrRNAs) and of varying lengths. They can include primary transcripts or processed forms. For example, tracrRNAs (as part of a single-guide RNA or as a separate molecule as part of a two- molecule gRNA) may comprise, consist essentially of, or consist of all or a portion of a wild type tracrRNA sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild type tracrRNA sequence). Examples of wild type tracrRNA sequences from S. pyogenes include 171-nucleotide, 89-nucleotide, 75 -nucleotide, and 65-nucleotide versions. See, e.g., Deltcheva et al. (2011) Nature 471(7340):602-607; WO 2014 / 093661, each of which is herein incorporated by reference in its entirety for all purposes. Examples of tracrRNAs within single-guide RNAs (sgRNAs) include the tracrRNA segments found within +48, +54, +67, and +85 versions of sgRNAs, where “+n” indicates that up to the +n nucleotide of wild typetracrRNA is included in the sgRNA. See US 8,697,359, herein incorporated by reference in its entirety for all purposes.

[0120] The percent complementarity between the DNA-targeting segment of the guide RNA and the complementary strand of the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%). The percent complementarity between the DNA-targeting segment and the complementary strand of the target DNA can be at least 60% over about 20 contiguous nucleotides. As an example, the percent complementarity between the DNA-targeting segment and the complementary strand of the target DNA can be 100% over the 14 contiguous nucleotides at the 5’ end of the complementary strand of the target DNA and as low as 0% over the remainder. In such a case, the DNA-targeting segment can be considered to be 14 nucleotides in length. As another example, the percent complementarity between the DNA-targeting segment and the complementary strand of the target DNA can be 100% over the seven contiguous nucleotides at the 5’ end of the complementary strand of the target DNA and as low as 0% over the remainder. In such a case, the DNA-targeting segment can be considered to be 7 nucleotides in length. In some guide RNAs, at least 17 nucleotides within the DNA-targeting segment are complementary to the complementary strand of the target DNA. For example, the DNA-targeting segment can be 20 nucleotides in length and can comprise 1, 2, or 3 mismatches with the complementary strand of the target DNA. In one example, the mismatches are not adjacent to the region of the complementary strand corresponding to the protospacer adjacent motif (PAM) sequence (i.e., the reverse complement of the PAM sequence) (e.g., the mismatches are in the 5’ end of the DNA-targeting segment of the guide RNA, or the mismatches are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs away from the region of the complementary strand corresponding to the PAM sequence).

[0121] The protein-binding segment of a gRNA can comprise two stretches of nucleotides that are complementary to one another. The complementary nucleotides of the protein-binding segment hybridize to form a double-stranded RNA duplex (dsRNA). The protein-binding segment of a subject gRNA interacts with a Cas protein, and the gRNA directs the bound Cas protein to a specific nucleotide sequence within target DNA via the DNA-targeting segment.

[0122] Guide RNAs can include modifications or sequences that provide for additional desirable features (e.g., modified or regulated stability; subcellular targeting; tracking with afluorescent label; a binding site for a protein or protein complex; and the like). That is, guide RNAs can include one or more modified nucleosides or nucleotides, or one or more non- naturally and / or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. Examples of such modifications include, for example, a 5’ cap (e.g., a 7-methylguanylate cap (m7G)); a 3’ polyadenylated tail (i.e., a 3’ poly(A) tail); a riboswitch sequence (e.g., to allow for regulated stability and / or regulated accessibility by proteins and / or protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (i.e., a hairpin); a modification or sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like); a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, and so forth); a modification or sequence that provides a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like); and combinations thereof. Other examples of modifications include engineered stem loop duplex structures, engineered bulge regions, engineered hairpins 3’ of the stem loop duplex structure, or any combination thereof. See, e.g., US 2015 / 0376586, herein incorporated by reference in its entirety for all purposes. A bulge can be an unpaired region of nucleotides within the duplex made up of the crRNA-like region and the minimum tracrRNA- like region. A bulge can comprise, on one side of the duplex, an unpaired 5'-XXXY-3' where X is any purine and Y can be a nucleotide that can form a wobble pair with a nucleotide on the opposite strand, and an unpaired nucleotide region on the other side of the duplex.

[0123] Guide RNAs can comprise modified nucleosides and modified nucleotides including, for example, one or more of the following: (1) alteration or replacement of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (2) alteration or replacement of a constituent of the ribose sugar such as alteration or replacement of the 2’ hydroxyl on the ribose sugar (an exemplary sugar modification); (3) replacement (e.g., wholesale replacement) of the phosphate moiety with dephospho linkers (an exemplary backbone modification); (4) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (5) replacement ormodification of the ribose-phosphate backbone (an exemplary backbone modification); (6) modification of the 3’ end or 5’ end of the oligonucleotide (e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap, or linker (such 3’ or 5’ cap modifications may comprise a sugar and / or backbone modification); and (7) modification or replacement of the sugar (an exemplary sugar modification). Other possible guide RNA modifications include modifications of or replacement of uracils or poly-uracil tracts. See, e.g., WO 2015 / 048577 and US 2016 / 0237455, each of which is herein incorporated by reference in its entirety for all purposes. Similar modifications can be made to Cas-encoding nucleic acids, such as Cas mRNAs. For example, Cas mRNAs can be modified by depletion of uridine using synonymous codons.

[0124] Chemical modifications such at hose listed above can be combined to provide modified gRNAs and / or mRNAs comprising residues (nucleosides and nucleotides) that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase. In one example, every base of a gRNAis modified (e.g., all bases have a modified phosphate group, such as a phosphorothioate group). For example, all or substantially all of the phosphate groups of a gRNA can be replaced with phosphorothioate groups. Alternatively, or additionally, a modified gRNA can comprise at least one modified residue at or near the 5’ end. Alternatively, or additionally, a modified gRNA can comprise at least one modified residue at or near the 3’ end.

[0125] Guide RNAs can be provided in any form. For example, the gRNA can be provided in the form of RNA, either as two molecules (separate crRNA and tracrRNA) or as one molecule (sgRNA), and optionally in the form of a complex with a Cas protein. The gRNA can also be provided in the form of DNA encoding the gRNA. The DNA encoding the gRNA can encode a single RNA molecule (sgRNA) or separate RNA molecules (e.g., separate crRNA and tracrRNA). In the latter case, the DNA encoding the gRNA can be provided as one DNA molecule or as separate DNA molecules encoding the crRNA and tracrRNA, respectively.

[0126] Alternatively, gRNAs can be prepared by various other methods. For example, gRNAs can be prepared by in vitro transcription using, for example, T7 RNA polymerase (see, e.g., WO 2014 / 089290 and WO 2014 / 065596, each of which is herein incorporated by reference in its entirety for all purposes). Guide RNAs can also be a synthetically produced molecule prepared by chemical synthesis. For example, a guide RNA can be chemically synthesized toinclude 2’-0-methyl analogs and 3’ phosphorothioate internucleotide linkages at the first three 5’ and 3’ terminal RNA residues.Guide RNA Target Sequences

[0127] Target DNAs for guide RNAs include nucleic acid sequences present in a DNA to which a DNA-targeting segment of a gRNA will bind, provided sufficient conditions for binding exist. Suitable DNA / RNA binding conditions include physiological conditions normally present in a cell. Other suitable DNA / RNA binding conditions (e.g., conditions in a cell-free system) are known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., Harbor Laboratory Press 2001), herein incorporated by reference in its entirety for all purposes). The strand of the target DNA that is complementary to and hybridizes with the gRNA can be called the “complementary strand,” and the strand of the target DNA that is complementary to the “complementary strand” (and is therefore not complementary to the Cas protein or gRNA) can be called “noncomplementary strand” or “template strand.”

[0128] The target DNA includes both the sequence on the complementary strand to which the guide RNA hybridizes and the corresponding sequence on the non-compl ementary strand (e.g., adjacent to the protospacer adjacent motif (PAM)). The term “guide RNA target sequence” as used herein refers specifically to the sequence on the non-complementary strand corresponding to (i.e., the reverse complement of) the sequence to which the guide RNA hybridizes on the complementary strand. That is, the guide RNA target sequence refers to the sequence on the noncomplementary strand adjacent to the PAM (e.g., upstream or 5’ of the PAM in the case of Cas9). A guide RNA target sequence is equivalent to the DNA-targeting segment of a guide RNA, but with thymines instead of uracils. As one example, a guide RNA target sequence for an SpCas9 enzyme can refer to the sequence upstream of the 5’-NGG-3’ PAM on the non-complementary strand. A guide RNA is designed to have complementarity to the complementary strand of a target DNA, where hybridization between the DNA-targeting segment of the guide RNA and the complementary strand of the target DNA promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided that there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. If a guide RNA is referred to herein as targeting a guide RNA target sequence, what is meant is that the guide RNA hybridizes to the complementary strand sequence of the target DNA that is the reverse complement of the guide RNA target sequence on the non-complementary strand.

[0129] A target DNA or guide RNA target sequence can comprise any polynucleotide, and can be located, for example, in the nucleus or cytoplasm of a cell or within an organelle of a cell, such as a mitochondrion or chloroplast. A target DNA or guide RNA target sequence can be any nucleic acid sequence endogenous or exogenous to a cell. The guide RNA target sequence can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory sequence) or can include both.

[0130] Formation of a CRISPR complex hybridized to a target DNA can result in cleavage of one or both strands of the target DNA within or near the region corresponding to the guide RNA target sequence (i.e., the guide RNA target sequence on the non-complementary strand of the target DNA and the reverse complement on the complementary strand to which the guide RNA hybridizes). For example, the cleavage site can be within the guide RNA target sequence (e g., at a defined location relative to the PAM sequence). The “cleavage site” includes the position of a target DNA at which a Cas protein produces a single-strand break or a double-strand break. The cleavage site can be on only one strand (e.g., when a nickase is used) or on both strands of a double-stranded DNA. Cleavage sites can be at the same position on both strands (producing blunt ends; e.g., Cas9)) or can be at different sites on each strand (producing staggered ends (i.e., overhangs); e.g., Cpfl). The guide RNA target sequence can also be selected to minimize off- target modification or avoid off-target effects (e.g., by avoiding two or fewer mismatches to off- target genomic sequences).

[0131] Interactions between integrated exogenous DNA and a host genome can limit the reliability and safety of integration and can lead to overt phenotypic effects that are not due to the targeted genetic modification but are instead due to unintended effects of the integration on surrounding endogenous genes. For example, randomly inserted transgenes can be subject to position effects and silencing, making their expression unreliable and unpredictable. Likewise, integration of exogenous DNA into a chromosomal locus can affect surrounding endogenous genes and chromatin, thereby altering cell behavior and phenotypes. Safe harbor loci include chromosomal loci where transgenes or other exogenous nucleic acid inserts can be stably and reliably expressed in all tissues of interest without overtly altering cell behavior or phenotype (i.e., without any deleterious effects on the host cell). See, e.g., Sadelain et al. (2012) Nat. Rev. Cancer 12:51-58, herein incorporated by reference in its entirety for all purposes. For example, the safe harbor locus can be one in which expression of the inserted gene sequence is notperturbed by any read-through expression from neighboring genes. For example, safe harbor loci can include chromosomal loci where exogenous DNA can integrate and function in a predictable manner without adversely affecting endogenous gene structure or expression. Safe harbor loci can include extragenic regions or intragenic regions such as, for example, loci within genes that are non-essential, dispensable, or able to be disrupted without overt phenotypic consequences.

[0132] Such safe harbor loci can offer an open chromatin configuration in all tissues and can be ubiquitously expressed during embryonic development and in adults. See, e.g., Zambrowicz et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:3789-3794, herein incorporated by reference in its entirety for all purposes. In addition, the safe harbor loci can be targeted with high efficiency, and safe harbor loci can be disrupted with no overt phenotype. Examples of safe harbor loci includePatent Nos. 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526; and US Patent Publication Nos. 2003 / 0232410; 2005 / 0208489; 2005 / 0026157; 2006 / 0063231; 2008 / 0159996; 2010 / 00218264; 2012 / 0017290; 2011 / 0265198; 2013 / 0137104; 2013 / 0122591; 2013 / 0177983; 2013 / 0177960; and 2013 / 0122591, each of which is herein incorporated by reference in its entirety for all purposes.

[0133] In a specific example, a safe harbor locus is a locus within the genome wherein a gene may be inserted without significant deleterious effects on the embryo (e.g., without causing apoptosis, necrosis, and / or senescence, as compared to a control embryo). The safe harbor locus can allow overexpression of an exogenous gene (e.g., METTL7A)' without significant deleterious effects on the embryo (e.g., without causing apoptosis, necrosis, and / or senescence, as compared to a control embryo). A desirable safe harbor locus may be one in which expression of the inserted gene sequence is not perturbed by read-through expression from neighboring genes. The safe harbor may be, for example, a bovine safe harbor or a human safe harbor.

[0134] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number, if applicable. Likewise, ifdifferent versions of a publication, website, or the like are published at different times, the version most recently published at the effective fding date of the application is meant, unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.BRIEF DESCRIPTION OF THE SEQUENCES

[0135] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and one letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5' end of the sequence and proceeding forward (z.e., from left to right in each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. When a nucleotide sequence encoding an amino acid sequence is provided, it is understood that codon degenerate variants thereof that encode the same amino acid sequence are also provided. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward i.e ., from left to right in each line) to the carboxy terminus.Table 1. Description of SequencesEXAMPLESExample 1. METTL7A improves the developmental potential of bovine IVP embryos.

[0136] Using CRISPR / Cas9, we created bovine METTL7AKOzygotes to investigate its role during pre-implantation development. There were no significant differences on cleavage rate (n = 12, p = 0.0686) and blastocyst formation (n = 6, p = 0.0729) between the knockout and control group (Fig. 5, Panels A and B). Mining of RNA-seq datasets also showed that METTL7A is barely expressed and translated across bovine oocytes and pre-implantation embryos derived in vitro (Fig. 5, Panel C), suggesting that METTL7A is dispensable for bovine pre-implantation development.

[0137] To explore the biological function of METTL7A during embryogenesis, we microinjected exogenous METTL 7 A mRNA into zygotes and evaluated the effect of overexpression (OE) of METTL7A during bovine pre-implantation development. While there is no difference in cleavage rate between METTL7AOEand the control group (79.29% vs. 81.42%; N= 980, n =29 for the treatment group; p = 0.4503), a 14.32% increase in blastocyst formation rate was observed in METTL7AOEcompared to controls (54.96% vs. 40.64%; N= 335, n =11 for the treatment group; p = 0.0102) (Fig. 1, Panels A and B), indicating a beneficial role of METTL7A for bovine pre-implantation development. Additionally, METTL7AOEblastocysts had a normal differentiation into inner cell mass (ICM) and trophectoderm (TE), as assessed by immunostaining analysis of SOX2 and CDX2 (Fig. 1, Panels C and D), respectively. Notably, the number of TE cells was significant higher in the METTL7AOEblastocysts compared to the control group (134 vs. 87; n = 5 p = 0.0198), while ICM cell number was not different (29 vs. 27.6; n = 5; p = 0.5548), resulting in higher TE / ICM ratio in METTL7AOEblastocysts compared to control (4.81 vs. 3.16, p = 0.0699) (Fig. 1, Panels E, F, and G).

[0138] To further determine the viability of METTL7AOEembryos and if they can establish successful pregnancy, we transferred either METTL7AOEor IVF embryos at morula stage to recipient cows and flushed them out on embryonic day 12 (E12) for analysis. We found METTL7AOEembryos displayed normal morphology and lineage differentiation similar to the control group (Fig. 1, Panels H and J) and initiated maternal recognition of pregnancy as indicated by a comparable serum INF-tau level in the surrogate mothers as IVF embryo transfers (n = 2, Fig. 1, Panel I).

[0139] These results demonstrated that METTL7A promotes the developmental potential of bovine pre-implantation embryos by facilitating TE lineage development, and that bovine METTL7AOEembryos produce normal pregnancy through conceptus elongation following embryo transfer to recipients.Example 2. METTL7A modulates expression of genes involved in mitochondrial functions during bovine pre-implantation development.

[0140] To understand the effect of METTL7A on gene expression in embryos and embryonic (ICM and TE) lineages, we performed RNA sequencing (RNA-seq) analysis on METTL7AOEand control embryos at 2-, 8-cell and blastocyst stage. ICM and TE were separated by micromanipulation procedures, which were confirmed by immunostaining analysis of lineage markers SOX2 and CDX2, respectively (Fig. 6, Panels A and B). Pearson correlation and principal component analysis of transcriptomic data indicated consistent values between biological replicates across developmental stage (Fig. 2, Panels A and B). While the transcriptomes of both METTL7AOEand control embryos were distinct across developmental stages, they appeared to cluster together within the same stage with more notable differences in 2 and 8-cell embryos than blastocysts (ICM and TE) (Fig. 2, Panels A and B).

[0141] In 2-cell embryos, we found 139 and 632 genes to be up- and down-regulated, respectively, (FDR P value < 0.05, |log2FC| >1) in METTL7AOEcompared to control embryos (Fig. 2, Panel C). The most up-regulated genes include METTL 7A, LOC781439, BTG2, STC1, ZSCAN5B, and PLA2G7 (Fig. 2, Panel C). Of note, METTL7A and LOC781439 (a pseudogene with truncated sequence of METTL7A) were the most regulated genes with log2FC > 11, confirming the overexpression c METTL7A. Among them, BTG2 has been reported to destabilize mRNA, while ZSCAN5B is associated to embryonic genome activation through modulating mitotic progression and safeguarding DNA damage response. Given the importance of maternal mRNA clearance for embryonic development, METTL7A overexpression may facilitate embryonic genome activation via downstream effectors like BTG2 and ZSCAN5B. Additionally, STC1 is hypoxia-responsive and promotes lipid metabolism, and its abundance is positively associated with all Ovum Pick Up-In Vitro Production (OPU-IVP) scores. Similarly, PLA2G7 (also known as LDL-PLA2) regulates phospholipid catabolism during inflammationand oxidative stress responses. The up-regulation of STC1 and PLA2G7 in METTL7AOEembryos suggested that METTL7A can alleviate oxidative stress in early bovine embryos.

[0142] At the 8-cell stage, 112 and 1,436 genes were up- and down-regulated, respectively, in METTL7AOEcompared to control embryos (Fig. 2, Panel D) Similarly, METTL7A and LOC781439 remained the top two up-regulated genes with lo 2FC > 11, indicating METTL 7 A overexpression pertains to the 8-cell stage. Other top up-regulated genes included KRT23, TARP, CXCL5, and SLC4A10, with known biological roles in promoting proliferation, DNA damage response, tumor progression, and pH balance, respectively. Compared to 2-cell stage, there were significantly more down-regulated genes in METTL7AOEembryos at the 8-cell stage. Only one gene had log2FC < -5 at the 2-cell stage while 401 genes showed log2FC < -5 at the 8-cell stage (Fig. 2, Panels C and D). Most of the top down-regulated genes in 8-cell embryos were associated with various stress responses, such as MAP1LC3C (as known as LC3C) and OASJX that are responsible for antibacterial and antiviral response.

[0143] At the blastocyst stage, we observed a larger number of genes differentially expressed in TE than ICM (up-regulated: 1,260 vs. 581; down-regulated: 539 vs. 239) associated with METTL7A overexpression (Fig. 2, Panels E and F). However, METTL7A was no longer up- regulated in the blastocysts (both TE and ICM), indicating that the observed transcriptomic changes were not directly caused by overexpression of METTL7A but rather from an altered gene expression cascade induced from cleavage stages.

[0144] Gene ontology (GO) analysis indicated overexpression (XMETTL7A suppressed genes involved in mitochondrial stress and functions among 8-cell and blastocyst (ICM and TE) stage embryos (Fig. 2, Panels G, H, and I). On the contrary, the up-regulated genes resulting from overexpression (XMETTL7A were involved in blastocyst formation at 8-cell stage, tissue development in ICM cells, and voltage-gated potassium and cation channel activities in TE cells, respectively (Fig. 2, Panels G, H, and I). Given that stress responses demand high energy consumption provided by mitochondria, the RNA-seq results suggested that overexpression of METTL7A shifts the paradigm of energy expenditure to favor bovine embryonic development.

[0145] Additionally, genes were found to be precisely modulated in the presence of METTL7A by comparing datasets between stages. For example, there is an earlier activation and up-regulation of HAND 1 observed at the 2-cell and the 8-cell embryos. HAND1 is essential for trophoblast lineage differentiation and development. However, at the blastocyst stage, theexpression of HAND! remain unchanged, coinciding with the termination of METTL7A overexpression in this stage.Example 3. METTL7A reduces mitochondrial stress and decreases superoxide levels of bovine pre-implantation embryos.

[0146] Given mitochondrial stress is precisely regulated in embryos and is associated with embryo competence. Given that the mitochondria-related pathways are among the top regulated in METTL7AOEembryos at 2-, 8-cell and blastocyst stage, we next sought to determine the effect of exogenous METTL7A on embryonic cell mitochondrial stress. We introduced a 6x His Tag before the stop codon of METTL7A due to the lack of a suitable bovine METTL7A antibody (Fig. 6, Panel C and Table 2) and established a lineage tracking system by 2-cell embryo microinjection. The intracellular localization of the expressed METTL7A-6xHis fusion protein was evaluated 12 hours after injecting the in vitro transcribed mRNAs into one of the blastomeres at the 2-cell stage (Fig. 3, Panel A). We confirmed that METTL7A was uniformly distributed into the cytoplasm, with no enrichment in particular organelles (Fig. 3, Panel B). We found that METTL7A-positive blastomeres progressed through one or two cell cycles within 12 hours, whereas METTL7A-negative blastomeres were arrested with condensed nuclei and degraded cytoskeleton (Fig. 3, Panel B), indicating an apoptotic cell fate. Moreover, METTL7A-negative blastomeres exhibited activation and nuclear translocation of HIF-la (Fig.3, Panel B), a marker of mitochondrial adaptation to oxidative stress. These results suggested that METTL7A can protect IVF embryos from mitochondrial stress, which was further supported by the presence of attenuated mitochondrial respiratory chain activities in METTL7AOEembryos (10 blastocysts / replicate, n = 6,p = 0.0142) (Fig. 3, Panels C-E) and was consistent with downregulation of mitochondrial pathways in METTL7AOEembryos (Fig. 2, Panels G-I)

[0147] To further delineate the mitochondrial stress relief conferred by METTL7A, we measured a cause of oxidative stress, reactive oxygen species (ROS), in METTL7AOEand control embryos. As expected, superoxide levels were reduced in METTL7AOEembryos compared to control (8-cell stage, p = 0.0454; blastocyst stage, p = 0.0182), as measured by the MitoSox green assay (Fig. 3, Panels F, G, and H). In accordance with lower superoxide levels, the levels of the intracellular antioxidant glutathione in its reduced form (GSH) also decreased dramatically (Fig. 3, Panels I and J) in METTL7AOEembryos compared to control (8-cellstage, p = 0.0023; blastocyst stage, p = 0.0007), indicating active reduction reactions in METTL7AOEembryos.

[0148] Together, these results indicated that METTL7A alleviates mitochondrial stress and prevents apoptosis during bovine pre-implantation embryo development.Example 4. METTL7A attenuates DNA damage and promotes cell cycle progression.

[0149] ROS are also genotoxic, prompting us to evaluate the effect of overexpression of METTL7A on embryonic cell DNA damage. DNA damage occurred in normal IVP embryos (Fig. 4, Panel A), consistent with previous findings. Using the same blastomere injection approach, we found METTL7A-negative blastomeres had a higher level of DNA damage, particularly at the 4-cell stage, as measured by yH2A.X staining (Fig. 4, Panel A). At the blastocyst stage, coincided with higher blastocyst rate, a lower level of DNA damage was observed in METTL7AOEembryos compared to control (p = 0.0288) (Fig. 4, Panels B and C). These results demonstrated that METTL7A reduces embryonic cell oxidative stress and DNA damage, thereby promoting the developmental potential of bovine IVP embryos.

[0150] Given that DNA damage can lead to the delayed cell cycles and impaired embryo development, we further analyzed p-Chkl, an essential marker for the DNA damage checkpoint and control of mitotic entry, in the METTL7A modulated embryos. We found that p-Chkl is significantly up-regulated in blastomeres 12 hours post-injection of METTL7A-6xHis fusion protein (Fig. 4, Panel D). However, at the blastocyst stage, the percentage of proliferating cells did not differ between METTL7AOEand control embryos (Fig. 4, Panels E and F), consistent with our previous observation that a lack of exogenous METTL7A persists into blastocyst stage embryos (Fig. 2, Panels E and F).

[0151] In summary, our results demonstrated that METTL7A ameliorates DNA damage by reducing ROS levels and enhancing DNA damage repair through p-Chkl , which promotes the ‘error-free’ cell cycle progression and pre-implantation embryo development.Materials and MethodsAnimal care and use

[0152] Bovine peri -implantation embryos were collected from non-lactating, 3-y ear-old crossbreed (Bos taurus x Bos indicus) cows. The experiments were conducted under animal useprotocols (202300000191) approved by the Institutional Animal Care and Use Committee of the University of Florida. All cows were housed in open pasture, and under constant care of the farm staff.Bovine oocytes and in vitro embryo production

[0153] Germinal vesicle stage oocytes (GV oocytes) were collected as cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries. In vitro maturation was conducted using BO-IVM medium (IVF Bioscience, Falmouth, UK) for 22-23 hours at 38.5°C with 6% CO2 to collect Mil oocytes. Cryopreserved semen from a Holstein bull with proven fertility was prepared with BO-SemenPrep medium (IVF Bioscience, Falmouth, UK) and added to drops containing COCs with a final concentration of 2 x 106spermatozoa / ml for in vitro fertilization. Gametes were co-incubated under 38.5°C and 6% CO2. After 10 hours (microinjected embryo experiments) or 16 hours (non-microinjected embryo experiments) in BO-IVF medium (IVF Biosciences, Falmouth, UK), IVF embryos were denuded from cumulus cells by vortexing for 5 min in BO-Wash medium (IVF Bioscience, Falmouth, UK) and cultured up to 7.5 d in BO-IVC medium (IVF Biosciences, Falmouth, UK) at 38.5°C, 6% CO2, and 6% O2. Different developmental stage embryos were then evaluated under light microscopy following embryo grade standards of the International Embryo Technology Society.In vitro transcription ofMETTL7A

[0154] Total RNA was extracted from a pool of bovine IVF embryos, embryonic stem cells, and trophoblast stem cells, followed by first strand cDNA synthesis using SuperScript™ IV VILO™ Master Mix (Thermo Fisher Scientific, Waltham, MA). Primers were designed based on the current genome annotation (ARS-UCD2.0; National Center for Biotechnology Information) to include 5’- and 3’-UTR regions o METTL7A (Table 2). PCR was conducted using Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA) with an initial denaturation step at 98°C for 30 seconds followed by 30 cycles at 98°C for 10 seconds, annealing at 58°C for 30 seconds and extension at 72°C for 30 seconds and a final extension at 72°C for 2 minutes. The purified PCR products were served as DNA template for in vitro transcription using HiScribe® T7 ARCA mRNA Kit with tailing following manufacture’s instruction. The yield and integrity of resulting mRNA were assessed using Qubit 4 (Thermo Fisher Scientific, Waltham, MA) and Tapestation 4150 (Agilent Technologies, Santa Clara, CA).

[0155] To generate METTL7A-6xHis mRNA, two pairs of primers (Table 2) were designed to produce PCR fragments with an overlapping region which contains 6xHis sequence before the stop codon o METTL7A. The two fragments were then assembled using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA), followed by in vitro transcription using using HiScribe® T7 ARCA mRNA Kit (New England Biolabs, Ipswich, MA). To overexpress METTL7A, in vitro transcribed mRNAs (IVT-mRNAs) were microinjected into presumptive zygotes or one blastomere of the 2-cell embryo at final concentration of 10 ng / uL.Table 2. Primer design for in vitro transcription of METTL7A and validation.Immunofluorescence and Data Analysis

[0156] Bovine embryos and embryonic cells were fixed in freshly made 4% paraformaldehyde (Electron Microscopy Science, Hatfield, PA) at room temperature for 15 minutes followed by permeabilization in 1% Triton X-100 (Sigma, Burlington, MA) for 20 minutes and blocking at room temperature for 1 hour in 0.1% Triton X-100, 0.1M glycine, 2.5% BSA (Sigma, Burlington, MA) and 2.5% corresponding serum from the host where the secondary antibodies were derived. Samples were then incubated with primary antibodies at 4°C overnight. After three washes in 0.1% Triton X-100 and 0.1% polyvinylpyrrolidone (PVP;Sigma, Burlington, MA) in Dulbecco's phosphate buffered saline (DPBS; Thermo Fisher Scientific, Waltham, MA), secondary antibodies were added and incubated at room temperature for 1 hour followed by three washes and mounting on the slide. Confocal images were taken with Olympus IX81-DSU (CGRC, University of Florida) and analyzed with Imaged.Embryo Transfer

[0157] E6 morulas were transferred to recipient cows following synchronization with initial intramuscular injection of gonadotropin-releasing hormone (Fertagyl; Merck, Rahway, NJ), standard 7-day vaginal controlled internal drug release (EAZLBREED CIDR; Zoetis, Parsippany-Troy Hills, NJ) of progesterone, one does of prostaglandin (Lutalyse; Zoetis, Parsippany-Troy Hills, NJ) upon CIDR removal and another dose of gonadotropin-releasing hormone 48 hours after CIDR removal. A cohort of 10 morulas from control or treatment group were loaded into 0.5 mL straws in prewarmed Holding Medium (ABT 360, Pullman, WA) and transferred non-surgically to the uterine horn ipsilateral to corpus luteum as detected by transrectal ultrasound. Embryos were recovered by standard non-surgical flushing with lactated ringer’s solution (ICU Medical, San Clemente, CA) supplemented with 1% fetal bovine serum on embryonic day 12. After flushing, all surrogate cows were given one dose of prostaglandin.Interferon-Tau Assay

[0158] Blood samples from recipients were collected from the coccygeal vein using serum separator tubes on the day of flushing, and immediately stored in refrigerator before centrifugation for 15 minutes at 1000 x g. Serum IFNT level was measured with Bovine Interferon-Tau ELISA Kit (CUSABIO, Houston, TX) per manufacture’s instruction. Briefly, 100 pL standard or sample were added to each well of 96-well plate provided in the kit and incubated for 2 hours at 37°C. Liquid was withdrew and 100 pL biotin-antibody was added to each well, followed by 1 hour incubation at 37°C. The solution was discarded, and the wells were washed three times with 200 pL Wash Buffer. To remove any remaining Wash Buffer in the wells, the plate was inverted and placed on clean paper towel for 1 minute. 100 pL HRP-avidin was then added to each well and incubated for 1 hour at 37°C followed by five times of washes. For signal detection, 90 pL TMB Substrate was added and incubated for 20 minutes at 37°C avoiding light. After incubation, 50 pL Stop Solution was added to each well while gently shaking the plate to ensure thorough mixing. The plate was measured using a colorimetric microplate reader set to 450 nm.Separation of TE and ICM

[0159] E7.5 blastocysts with zona pellucida were used for TE / ICM dissociation following previously published protocol (see, e.g., Wang et al. (2023) Cell Rep. 2023: 112439; herebyincorporated by reference in its entirety for all purposes). Briefly, 0.25% trypsin (Thermo Fisher Scientific, Waltham, MA) was continuously injected into the blastocysts until a small mass of cells was slowly washed out of the zona pellucida. The cell masses were washed three times with 0.1% PVP and immediately transferred to -80°C until further use or fixed for staining.RNA sequencing analysis

[0160] Five 2- or 8-cell embryos were pooled in each replicate and TE and ICM cell clumps from 5 blastocysts were pooled after separation for RNA-seq library preparation. Embryos and cells were used directly for library preparation without RNA extraction following manufacturers’ instructions. Briefly, SMART-Seq v4 Ultra Low Input RNA kit (Takara, Mountain View, CA) was used for cDNA synthesis and amplification. Library preparation was conducted using Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA). The libraries were subject to size selection with 0.6x AMPure XP bead wash (Beckman Coulter, Indianapolis, IN). The concentration of RNA-seq libraries was determined with a Qubit high sensitivity dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA). Pooled indexed libraries were then sequenced on the Illumina NovaSeq 6000 platform with 150-bp paired-end reads.

[0161] Multiplexed sequencing reads that passed filters were trimmed to remove low-quality reads and adaptors by Trim Galore (version 0.6.7). The quality of reads after filtering was assessed by FastQC, followed by alignment to the bovine reference genome by HISAT2 (version 2.2.1) with default parameters. The output SAM files were converted to BAM files and sorted using SAMtools6 (version 1.14). Read counts of all samples were quantified using featureCounts (version 2.0.1) with the bovine genome as a reference and were adjusted to provide counts per million (CPM) mapped reads. Pearson correlation and Principal Component analysis were performed with R (a free software environment for statistical computing and graphics). Differentially expressed genes were identified using edgeR in R. Genes were considered differentially expressed when they provided a false discovery rate of <0.05 and fold change >2. ClusterProfiler was used to reveal the Gene Ontology and KEGG pathways in R.Western Blot and Data Analysis

[0162] Ten E7.5 blastocysts were washed three times in 0.1% PVP and pooled with approximately 5 pL medium carryover in each replicate. Samples were first heated with 5 pL SDS gel-loading buffer at 95°C for 5 minutes followed by a quick spin down, and then loadedonto 10% Tris-Glycine Mini Protein Gels (Thermo Fisher Scientific, Waltham, MA). Western blot electrophoresis was set to 100 volts for 2 hours. Proteins were transferred from gel to PVDF membrane with iBlot 3 Western Blot Transfer Device (Thermo Fisher Scientific, Waltham, MA). After transfer, the membrane was washed with 25 mL Tris buffered saline (TBS) for 5 minutes at room temperature followed by blocking with 2.5% BSA and 2.5% corresponding serum, from the host where the secondary antibodies were derived, for Ih at room temperature. The membrane was washed three times for 5 minutes each with 15 mL of TBST (0.1% Tween-20 in TBS). Primary antibodies were added in 10 mL dilution buffer (5% w / v BSA in TBST) and incubated with membrane with gentle agitation overnight at 4°C followed by three times of washes with TBST. Secondary antibodies were added in 10 mL of blocking buffer and incubated with membrane with gentle agitation for 1 hour at room temperature. Membrane was washed three times before proceeded with signal detection. Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA) was added to the membrane and incubated for 1 minute. Excessive solution was removed before imaging using iBright CL1500 System (Thermo Fisher Scientific, Waltham, MA).Superoxide Assay

[0163] MitoSOX Green (MSG) (Thermo Fisher Scientific, Waltham, MA) reagent stock was prepared by dissolving the contents of the vial in 10 pL of anhydrous DMF, which is stable for one day. To make a working solution, 10 pL of ImM stock solution was added to HEPES- TALP. 200 pL of working solution was added to one well of p-Slide (Ibidi, Fitchburg, WI). Live embryos were taken out from culture and washed quickly with HEPES-TALP followed by incubation in MSG working solution for 30 minutes at 38.5°C, 6% CO2. After incubation, embryos were washed three times with warm buffer and confocal images were taken within 2 hours of staining. Fluorescence intensity was analyzed with Imaged and Prism 9 (GraphPad, La Jolla, CA). Two-tailed student’s t-test was used for statistical analysis.GSH Assay

[0164] To measure the level of glutathione in reduced form (GSH), ten 8-cell embryos or five E7.5 blastocysts were pooled in each replicate after washing briefly in 0.1% PVP / PBS. The level of reduced GSH was quantified indirectly by subtracting oxidized GSH (GSSG) from total GSH using commercial kit GSH / GSSG-Glo™ Assay (Promega, Madison, WI) following themanufacturer’s instructions. Relative luminescence over no cell control was analyzed with two- tailed student’ s t-test in Prism 9.

Claims

We claim:

1. A method for improving in vitro produced embryo competence comprising increasing METTL7A in an embryo.

2. The method of claim 1, wherein exogenous METTL7A protein or a fragment thereof is introduced into the embryo.

3. The method of claim 2, wherein the introducing comprises microinjection into a presumptive zygote or one blastomere of a two-cell embryo.

4. The method of claim 1, wherein a polynucleotide encoding METTL7A is introduced into the embryo.

5. The method of claim 4, wherein the polynucleotide is DNA.

6. The method of claim 4, wherein the polynucleotide is RNA.

7. The method of claim 4, wherein the introducing comprises:(i) microinjection of the polynucleotide into a presumptive zygote or one blastomere of a two-cell embryo; or(ii) administration of a gene therapy vector comprising the polynucleotide to a presumptive zygote or two-cell embryo.

8. The method of claim 7, wherein the polynucleotide is introduced via administration of a gene therapy vector, and wherein the gene therapy vector comprises:(i) a polynucleotide complex; or(ii) a viral vector.

9. The method of claim 8, wherein the gene therapy vector is a polynucleotide complex, and wherein the polynucleotide complex is a lipid nanoparticle comprising the polynucleotide and lipids.

10. The method of claim 8, wherein the gene therapy vector is a viral vector, and wherein the viral vector is selected from the group consisting of a retrovirus, an adenovirus, a herpes simplex virus, a pox virus, a vaccinia virus, a lentivirus, or an adeno-associated virus (AAV).

11. The method of claim 4, further comprising a nuclease agent.

12. The method of claim 11, wherein the nuclease agent comprises:(a) a zinc finger nuclease (ZFN);(b) a transcription activator-like effector nuclease (TALEN); or(c) (i) a Cas protein or a nucleic acid encoding the Cas protein; and(ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

13. The method of claim 4, wherein the polynucleotide is genomically integrated.

14. The method of claim 4, wherein the polynucleotide is extrachromosomal.

15. The method of claim 1, wherein the embryo is a mammalian embryo.

16. The method of claim 15, wherein the mammalian embryo is a bovine embryo.

17. The method of claim 15, wherein the mammalian embryo is a human embryo.

18. A composition comprising METTL7A protein for use in the method of claim 2.

19. A composition comprising a polynucleotide encoding METTL7A for use in the method of claim 4.

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