Transactivation of miR-202-5p by Steroidogenic Factor 1 (SF1) Induces Apoptosis in Goat Granulosa Cells by Targeting TGFβR2

MicroRNAs play key roles during ovary development, with emerging evidence suggesting that miR-202-5p is specifically expressed in female animal gonads. Granulosa cells (GCs) are somatic cells that are closely related to the development of female gametes in mammalian ovaries. However, the biological roles of miR-202-5p in GCs remain unknown. Here, we show that miR-202-5p is specifically expressed in GCs and accumulates in extracellular vesicles (EVs) from large growth follicles in goat ovaries. In vitro assays showed that miR-202-5p induced apoptosis and suppressed the proliferation of goat GCs. We further revealed that miR-202-5p is a functional miRNA that targets the transforming growth factor-beta type II receptor (TGFβR2). MiR-202-5p attenuated TGF-β/SMAD signaling through the degradation of TGFβR2 at both the mRNA and protein level, decreasing p-SMAD3 levels in GCs. Moreover, we verified that steroidogenic factor 1 (SF1) is a transcriptional factor that binds to the promoters of miR-202 and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) through luciferase reporter and chromatin immunoprecipitation (ChIP) assays. That contributed to positive correlation between miR-202-5p and CYP19A1 expression and estradiol (E2) release. Furthermore, SF1 repressed TGFβR2 and p-SMAD3 levels in GCs through the transactivation of miR-202-5p. Taken together, these results suggest a mechanism by which miR-202-5p regulates canonical TGF-β/SMAD signaling through targeting TGFβR2 in GCs. This provides insight into the transcriptional regulation of miR-202 and CYP19A1 during goat ovarian follicular development

Markhor-derived introgression of PAPSS2 confers high-altitude adaptability in Tibetan goats

Understanding the genetic mechanism of how animals adapt to extreme environments is fundamental to unriddle the relationship between molecular evolution and continuous climate change. After domestication, goats have developed strong adaptation to various environments, including harsh conditions at high-altitudes with low temperature and reduced oxygen concentrations. Here, we utilized 331 genomes of goats living at varying altitudes (high >3,000 m above sea level and low <1,200 m) as well as wild caprid species to precisely dissect genetic determinants underlying the adaptation to high altitudes on the Qinghai-Tibetan Plateau (QTP). Population genomic analyses combined with genome-wide association testing revealed that the PAPSS2 locus confers adaptability to high altitudes in Tibetan goats. Additionally, this gene harbours significant signatures of interspecies introgression from a wild caprid species, markhor (Capra falconeri). We further demonstrated a functional role for PAPSS2 in response to hypoxia using PAPSS2-deficient cells. In conclusion, our results suggest a hitherto unknown contribution of PAPSS2 to high-altitude adaptation and showed that interspecific introgression contributed to the adaptation of goats to the harsh conditions at the Tibetan Plateau

Additional file 1 of Generation of sheep with defined FecBB and TBXT mutations and porcine blastocysts with KCNJ5G151R/+ mutation using prime editing

Additional file 1: Fig S1. Editing efficiency in the generated lambs as shown by targeted deep sequencing. Fig. S2. Detection of potential off-target sites by Sanger sequencing in founder animals. Five potential off-target sites (Spacer OT1-OT5) were predicted by Cas-OFFinder in FecBB-edited lambs. Sanger sequencing was used to determine variations at predicted target sites for the five founder animals. Fig. S3. Detection of potential off-targeted sites by Sanger sequencing in founder animals. Nine potential off-target sites (Nick OT1-OT9) were predicted by Cas-OFFinder in FecBB-edited lambs. Sanger sequencing was used to determine variations at predicted target sites for the five founder animals. Fig. S4. Detection of potential off-targeted sites by Sanger sequencing in founder animals. A potential off-target site (Spacer OT1) was predicted by Cas-OFFinder in TBXT-edited lambs. Sanger sequencing was used to determine variations at predicted target sites for the three founder animals. Fig. S5. Detection of potential off-targeted sites by Sanger sequencing in founder animals. Fifteen potential off-target sites (Nick1 OT1-OT8 and Nick2 OT1-OT7) were predicted by Cas-OFFinder in TBXT-edited lambs. Sanger sequencing was used to determine variations at predicted target sites for the three founder animals. Fig. S6. Digital PCR of PE edited cell populations (Sp1-1, Sp1-2, and Sp1-3) and an unedited wild type control. (a, d, g, j, and m) chip view by calls. (b, c, e, f, h, I, k, l, n, and o) yellow: no amplification, red: VIC reporter dye signal (KCNJ5 WT probe), blue: FAM reporter dye signal (KCNJ5 G151R probe), green: FAM + VIC reporter dye signals. (b) Two-dimensional scatter plot of digital PCR SNP assay and (c) histogram from unedited wild type control cells. (e) Two-dimensional scatter plot of digital PCR SNP assay and (f) histogram from replicate Sp1-1. (h) Two-dimensional scatter plot of digital PCR SNP assay and (i) histogram from replicate Sp1-1 (repetition of dPCR chip). (k) Two-dimensional scatter plot of digital PCR SNP assay and (l) histogram from replicate Sp1-2. (n) Two-dimensional scatter plot of digital PCR SNP assay and (o) histogram from replicate Sp1-3. Fig. S7. Sanger sequencing of all ten porcine blastocysts shows a heterozygous p.G151R mutation next to the silent mutation of the DdeI restriction enzymatic digestion site. Fig. S8. Sanger sequencing of colonies after cloning of blastocyst PCR products into the pGEM-T Easy vector. (a) WT1b_3A_a3 and (b) WT1b_3A_a5 show overlapping sequences besides sequences containing both desired mutations and sequences matching to the wild-type reference sequence

Additional file 2 of Generation of sheep with defined FecBB and TBXT mutations and porcine blastocysts with KCNJ5G151R/+ mutation using prime editing

Additional file 2: Table S1. List of primers used for genotyping and amplifying PE3-targeted FecBB fragment in HEK293T cells. Table S2. Sequences of pegRNA and sgRNA used in human HEK293T cells. Table S3. Sequences of pegRNAs and sgRNAs used in sheep. Table S4. List of primers used for in vitro transcription. Table S5. List of primers for genotyping and amplifying PE3-targeted BMPR1B and TBXT fragments in newborn lambs. Table S6. List of predicted off-target sites for PE3-targeted BMPR1B. Table S7. List of predicted off-target sites for PE3-targeted TBXT. Table S8. List of primers for genotyping and amplifying predicted off-target site fragments in FecBB-edited sheep. Table S9. List of primers for genotyping and amplifying predicted off-target site fragments in TBXT-edited sheep. Table S10. Sequences of pegRNAs and sgRNAs used in pigs. Table S11. List of primers for genotyping and amplifying PE3-targeted KCNJ5 in porcine kidney fibroblasts and blastocysts. Table S12. Number of blastocysts generated per SCNT session using KCNJ5G151R/+ porcine kidney fibroblasts as donor cells

Additional file 3 of Generation of sheep with defined FecBB and TBXT mutations and porcine blastocysts with KCNJ5G151R/+ mutation using prime editing

Additional file 3: Table S1. The targeted deep sequencing-based analysis of the off-target site in BMPR1B-edited lambs. Table S2. The targeted deep sequencing-based analysis of the off-target sites in TBXT-edited lambs

Markhor-derived introgression of a genomic region encompassing PAPSS2 confers high-altitude adaptability in Tibetan goats

Understanding the genetic mechanism of how animals adapt to extreme conditions is fundamental to determine the relationship between molecular evolution and changing environments. Goat is one of the first domesticated species and has evolved rapidly to adapt to diverse environments, including harsh high-altitude conditions with low temperature and poor oxygen supply but strong ultraviolet radiation. Here, we analyzed 331 genomes of domestic goats and wild caprid species living at varying altitudes (high > 3000 m above sea level and low < 1200 m), along with a reference-guided chromosome-scale assembly (contig-N50: 90.4 Mb) of a female Tibetan goat genome based on PacBio HiFi long reads, to dissect the genetic determinants underlying their adaptation to harsh conditions on the Qinghai-Tibetan Plateau (QTP). Population genomic analyses combined with genome-wide association studies (GWAS) revealed a genomic region harboring the 3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2) gene showing strong association with high-altitude adaptability (P(GWAS) = 3.62 × 10(−25)) in Tibetan goats. Transcriptomic data from 13 tissues revealed that PAPSS2 was implicated in hypoxia-related pathways in Tibetan goats. We further verified potential functional role of PAPSS2 in response to hypoxia in PAPSS2-deficient cells. Introgression analyses suggested that the PAPSS2 haplotype conferring the high-altitude adaptability in Tibetan goats originated from a recent hybridization between goats and a wild caprid species, the markhor (Capra falconeri). In conclusion, our results uncover a hitherto unknown contribution of PAPSS2 to high-altitude adaptability in Tibetan goats on QTP, following interspecific introgression and natural selection