STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

No live animals were involved in this study. Bovine oocytes were produced from ovaries collected from slaughterhouse-derived bovine reproductive tracts and different stages of embryos were produced by in vitro fertilization. Because embryos cannot be sexed prior to experimental treatments, the analysis of pooled embryos at different developmental stages has ensured a representation of both male and female embryos.

METHOD DETAILS

Bovine oocytes and in vitro embryo production

Bovine ovaries were collected from slaughterhouse-derived bovine reproductive tracts, and cumulus-oocyte complexes (COCs) were aspirated from 3 to 5 mm follicles using an 18-gauge needle. Germinal vesicle stage oocytes (GV oocytes) were collected as cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries. Only COCs with a homogeneous cytoplasm and multiple cumulus cell layers were selected for in vitro maturation (IVM). Maturation was performed in BO-IVM medium (IVF Bioscience) at 38.5°C with 6% CO2 for 22–23 h. After maturation, cumulus cells were removed, and nuclear maturation was confirmed by assessing the presence of the first polar body under a light microscope. For in vitro fertilization (IVF), cryopreserved semen from Holstein bulls with proven fertility was processed using BO-SemenPrep medium (IVF Bioscience), and the final sperm concentration was adjusted to 2 × 106 spermatozoa/mL. Matured oocytes were co-incubated with sperm in fertilization medium at 38.5°C in 6% CO2 for 18 h. Following fertilization, presumptive zygotes were washed and cultured in synthetic oviduct fluid, BO-IVC medium (IVF Bioscience), in groups of approximately 30 embryos per 50-μL droplet overlaid with mineral oil. Embryos were incubated at 38.5°C in a benchtop incubator (WTA, Cravinhos, SP, Brazil) under a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 until day 7.5 post-fertilization. Cleavage rates were assessed on day 2, and blastocyst formation was evaluated on day 7.5 to determine developmental competence. This optimized embryo production system was used for downstream epitranscriptomic analysis, including m6A profiling using SAC-seq.

RNA isolation and SAC-seq

Total RNA was isolated from bovine oocytes and early embryos at different developmental stages using TRIsolution (Sigma-Aldrich) according to the manufacturer’s protocol, with modifications to optimize RNA recovery from small sample sizes. Approximately 200 oocytes or embryos were pooled per replicate for RNA extraction. Briefly, samples were lysed in TRIsolution, followed by phase separation with chloroform. The aqueous phase was collected, and RNA was precipitated with isopropanol, washed with ethanol, and resuspended in nuclease-free water. RNA purity and concentration were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and integrity was evaluated using an Agilent Bioanalyzer 2100 (Agilent Technologies). Only samples with RNA integrity numbers (RIN) above 7.0 were used for downstream analysis. Due to the low RNA input and the unavailability of a bovine-specific ribosomal RNA depletion kit, we proceeded without an rRNA depletion step. Instead, total RNA was directly subjected to SAC-seq (Selectively Allyl-Chemically Labeled m6A Sequencing) to achieve single-nucleotide resolution detection of m6A modifications. The RNA underwent selective allyl-chemical labeling of m6A sites, followed by reverse transcription using a modified primer designed to introduce mutation signatures indicative of m6A presence. cDNA libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (New England Biolabs) and sequenced on an Illumina NovaSeq 6000 platform to generate high-depth paired-end reads. Bioinformatic analysis was conducted using a custom computational pipeline to identify m6A sites and assess their dynamic regulation during early bovine embryonic development. The SAC-seq data were analyzed in the context of maternal RNA clearance and zygotic genome activation, providing novel insights into the epitranscriptomic mechanisms regulating bovine embryogenesis.

SAC-seq data analysis

Data preprocessing:

Raw sequencing data were trimmed using Cutadapt (v5.0) as described previously.48 Briefly, the 6-bp UMI sequence and following 5-bp RT tail were firstly trimmed from Read 2 and appended to the read name. The adapter sequences were trimmed from both ends of Read 1 and Read 2. Low quality bases were trimmed with parameter “-q 20”. Reads with a final length <15 bp were discarded.

Spike-in analysis and calibration curve for m6A stoichiometry estimation:

Trimmed reads were mapped to spike-in RNA using Bowtie249 (v2.2.8) and converted to FASTA format using Samtools50 (v1.18). The mapped reads were next assigned to each spike-in RNA sequence using blastn51 (v2.3.0+). The conversion rate of m6A/A base for each spike-in RNA was then calculated with a custom Python script. Calibration curves were then fit using stats.linregress function from Scipy package. The motifs with a negative slope were excluded from the following analyses.

Read mapping, mutation calling and identification of m6A sites:

Trimmed reads were mapped to bosTau9 (ARS_UCD1.3) using STAR52 (v2.7.9a). After removing the unmapped reads, PCR duplicates were identified and discarded based on UMI sequence and mapping coordinates by using UMI-Tools53 (v1.1.6). Variant calling was then performed by Samtools mpileup. All called variants were then filtered by the following criteria: 1) the corresponding loci covered by ≥ 10 reads in both treated and input libraries, 2) variants supported by ≥ 3 reads in each treated library, 3) average mutation ratio in input libraries is <0.01, 4) average mutation ratio in treated libraries is ≥0.05. The identified m6A sites were annotated by using variant-effect package (https://github.com/y9c/variant). To calculate the modification fraction, we then normalized the mutation ratio of each m6A site using the fitted calibration curve of the corresponding motif. The metagene profiles of detected m6A sites were generated using metaPlotR package.54

RNA-seq data analysis:

Raw sequencing reads were first trimmed as described above. We next mapped the trimmed reads to bosTau9 (ARS_UCD1.3) using STAR (v2.7.9a). Uniquely mapped reads were then assigned to genes using HTseq-count55 (v2.0.5) with parameters “-s no -t exon -m intersection-strict”. Normalization and differential gene expression analyses between adjacent time points were performed using edgeR package with a threshold of log2 fold change >1 and False discovery rate (FDR) < 0.05. Multiple testing correction was performed using the Benjamini–Hochberg method.56 Differentially expressed genes (DEGs) were clustered according to their expression dynamics across different time points using k-means clustering algorithm. To identify the sub-clusters of DEGs based on m6A modification, we first scaled the modification fractions of each m6A site across different time points. Next, we computed the average m6A modification fraction for each gene at different time points by aggregating the scaled modification fractions of all associated m6A sites. m6A marked DEGs were then clustered into sub-clusters based on their m6A modification dynamics across different time points using k-means clustering algorithm.

eTAM-seq analysis

RNA from oocytes and embryos were processed using a targeted amplicon eTAM-seq workflow. Cells were lysed in TRIzol reagent (Thermo Fisher Scientific, Cat# 15596026), and total RNA was purified using the Direct-zol RNA MiniPrep kit (Zymo Research, Cat# R2052) with on-column DNase I digestion provided with the kit. Purified RNA was subjected to a one-pot fragmentation and deamination reaction in deamination buffer (50 mM Tris-HCl pH 7.5, 25 mM KCl, 2.5 mM MgCl2, 2 mM DTT, and 10% glycerol) containing SUPERase·In RNase inhibitor (Thermo Fisher Scientific, Cat# AM2694). RNA was fragmented by heating at 94°C for 3 min followed by immediate cooling on ice. TadA deaminase was then added, and reactions were incubated at 53°C for 1 h followed by two additional incubations at 44°C for 1 h each. RNA was purified using the RNA Clean & Concentrator-5 kit (Zymo Research, Cat# R1015) to remove enzyme and reaction components. Reverse transcription was performed using SuperScript IV reverse transcriptase (Thermo Fisher Scientific, Cat# 18090010) with a multiplexed pool of gene-specific reverse primers and dNTP mix (New England Biolabs, Cat# N0447S). Target regions were amplified using NEBNext Ultra II Q5 Master Mix (New England Biolabs, Cat# M0544) with pooled forward and reverse primers. Indexed sequencing libraries were generated by a second PCR using universal P5 and P7 indexing primers (Takara Bio, Cat# 634413). Libraries were assessed using an Agilent High Sensitivity D1000 ScreenTape assay and purified with AM-Pure XP beads (Beckman Coulter, Cat# A63881) prior to sequencing on an Illumina platform.

Ribosome profiling data analysis

Bovine embryo ribosome dataset was downloaded from GSE196484.36 Raw sequencing data were first trimmed using Cutadapt (v5.0) to discard adapter sequences. Trimmed reads were then mapped to bosTau9 (ARS_UCD1.3) using STAR (v2.7.9a). Uniquely mapped reads were assigned to genes using HTseq-count (v2.0.5), and normalization was performed using edgeR package. F6 to F10 were used to calculate the expression levels of each gene in polysome-bound fraction at different time points.

m6A site editing using CRISPR PEmax

Bovine embryos were produced via in vitro fertilization (IVF) and collected at the zygote stage (16–18 h post fertilization) for prime editing. The optimized prime editing system, PEmax, was delivered via cytoplasmic microinjection of in vitro produced PEmax mRNA and a synthetic prime editing guide RNA (pegRNA) designed for the target locus. Microinjections were performed in BOWASH (IVF Bioscience) medium using a micromanipulator-equipped inverted microscope. Following microinjection, embryos were cultured in IVF-BIO IVC medium at 38.5°C under a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 until the blastocyst stage. Individual blastocysts were collected for genomic DNA (gDNA) extraction using the Red Extract-N-Amp kit (Sigma XNAT-100RXN) as previously described. Briefly, each embryo was lysed in 4 μL extraction buffer and 1 μL tissue preparation buffer at room temperature for 15 min, followed by heat inactivation at 95°C for 5 min. Samples were cooled to room temperature before adding 4 μL neutralization buffer, and DNA was stored at 4°C for no more than three days before subsequent PCR analysis. Editing efficiency was assessed via Sanger sequencing or next-generation sequencing (NGS), with quantification based on the percentage of successfully edited alleles.

Plasmid preparation, in vitro transcription, and microinjection

In vitro transcribed H2b:GFP RNA was prepared from a plasmid using the T3 mMessage mMachine Kit (Ambion), and it was used as an injection control. Plasmids pMSCV-dCasRx-ALKBH5 H204A (Mutant) and pMSCV-dCasRx-ALKBH5 (Wild-Type) were used as templates for in vitro transcription. A T7 overhang forward primer (Data S1) was used in PCR to amplify templates from the above plasmids for transcription. The PCR products were purified using the GeneElute PCR Purification Kit (NA1020, Sigma), followed by in vitro transcription and poly(A) tailing using the HiScribe® T7 ARCA mRNA Kit (New England Biolabs, Ipswich, MA). The RPL12 wild-type gene was subcloned into the pGEMHE vector (Addgene #105519) by replacing the TRIM21 sequence to generate the final construct pGEMHE_mEGFP_WTRPL12 (cloning performed by Azenta Life Sciences). This plasmid was linearized with PacI and purified using the GeneElute PCR Purification Kit (NA1020, Sigma) for use in in vitro transcription.

Zygote-stage embryos were microinjected with the in vitro transcribed H2B:GFP RNA in combination with CRISPR reagents. Microinjections were performed using a FemtoJet 4i microinjector (Eppendorf), an inverted microscope (Leica DMi8), and micromanipulators (Narishige). Embryos collected between 18 and 20 h post-IVF were used for injection. The injection solution contained 20 ng/μL of H2B:GFP RNA along with CRISPR components. Injected presumptive zygotes were cultured in 50 μL BO-IVC medium for further development, and embryo progression was monitored using a Leica DMI 6000 B inverted microscope.

Nascent protein detection

To visualize nascent protein synthesis, Click-iT HPG reagents (Invitrogen) were used. Embryos were incubated in 50 μM Click-iT HPG working solution in methionine-free medium for 2 h and processed according to the manufacturer’s protocol. Briefly, embryos were fixed with 3.7% formaldehyde in PBS for 15 min, followed by permeabilization with 0.5% Triton® X-100 for 20 min at room temperature. After three PBS washes, embryos were incubated with a Click-iT reaction cocktail containing Alexa Fluor-conjugated azide for 30 min (protected from light). Embryos were then mounted on slides using ProLong Gold Antifade Mountant with DAPI and scanned using a Leica SP5 confocal microscope. For the negative control, embryos were treated with cycloheximide (50 μg/mL) prior to HPG labeling.

RT–qPCR-based m6A identification

Site-specific m6A levels were quantified following previously published protocols57,58 with minor modifications. Total RNA was extracted from edited embryos (at least 10 embryos per replicate per group) using TRIzol (Sigma), with glycogen added to enhance RNA recovery. The RNA was treated with DNase I (Invitrogen) at 37°C for 30 min, followed by purification using the RNeasy MinElute Kit (Qiagen). Two reverse transcription reactions were prepared using either Bst DNA polymerase (NEB) or SuperScript III (Invitrogen), followed by PCR amplification and quantification exactly as described in the referenced protocol.57

RNA sequencing analysis of m6A-RPL edited embryos

Five to ten 4- or 8-cell embryos were pooled for RNA-seq library preparation. Embryos and cells were used directly for library preparation without RNA extraction following SMART-Seq v4 Ultra Low Input RNA kit (Takara, Mountain View, CA) manufacturer’s instructions. Pooled indexed libraries were then sequenced on the Illumina NovaSeq 6000 platform with 150-bp paired-end reads. RNA-seq analysis was conducted as described above.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical differences between pairs of datasets were analyzed by two-tailed unpaired t-tests. Values of p < 0.05 were considered statistically significant. Where applicable, all quantitative data were presented as the mean ± standard error or the mean. The number of biological replicates was indicated as ‘n’ in figure legends. Gene ontology enrichment analyses were performed using the goseq R package.59 The Benjamini-Hochberg (BH) procedure was used for multiple testing correction.