Search

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 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: Supplementary Fig. 2. The composition of gut microbiota in the MSTN-edited and wild type-groups. A. Bray-Curtis based PCoA of the gut microbiome in the GEM and WTM. The P-value was based on ANOSIM. The boxplot shows the discrete distribution of samples along the PC1 and PC2 axes. B. The histogram of the dominant phyla in the GEF, WTF, GEM and WTM. It excludes unclassified microbes. C. The significantly different phyla between the GEM and WTM. D. The significantly different genera between the GEM and WTM. E. The histogram of the dominant species in the GEF, WTF, GEM and WTM. It excludes unclassified microbes. F. The significantly different species between the GEM and WTM. G. LEfSe analysis of the GEM and WTM. The histogram shows the microbes that can best illustrate the difference between the GEM and WTM. The larger the LDA score, the greater the contribution of the corresponding microbe to the difference.

Additional file 1: Supplementary Fig. 1. The sequences of the second and third exons of MSTN based on Sanger sequencing.

Additional file 1: Table S1. Average cleavage rate and mutation rate of experimental groups. Table S2. Primers used for genotyping and amplifying Cas9/sgRNA-targeted MSTN fragment. Table S3. List of predicted off-target sites. Table S4. Growth parameters of MSTN-knockout and wild-type sheep. Table S5. sgRNA sequences and target sites. Table S6. Oligonucleotides used for generating sgRNA-expressing vectors for in vitro transcription. Table S7. Primers used for genotyping and amplifying predicted off-target site fragments. Table S8. Primers used for measuring MSTN expression level by real-time quantitative PCR (RT-qPCR). Description: The file contains the sequences of on- and off-target sites, primers, and oligonucleotides, as well as other relevant data.

Additional file 1: Figure S1. High-throughput sequencing outcomes for candidate genes linked with the sheep tail phenotype pattern. Outcomes of genomic a and transcriptomic b approaches. Figure S2. An example of coat color variation in sheep. Coat colors and markings in Shetland sheep breed. Poster copyright and published with the consent of the Shetland Sheep Society ( www.shetland-sheep.org.uk ). Figure S3. CRISPR/Cas9-generated ASIP knockout Chinese Merino sheep. a Badger-face with black body coat color. b Brown coat color with light ventral pigmentation. c Black-white spotted coat color. d Wild-type phenotype. Figure adapted from Zhang et al. [83]. Figure S4. Phenotypic variations of face coat color in sheep. A representative example from Chinese Tan sheep with varied face coat colors, including white, light brown spots, dark brown spots, brown, black spots, black marks, and black. Photos were taken by P.K. at Ningxia Tianyuan Tan Sheep Farm, Hongsibu, China. Figure S5. Phenotypic variations of nipple (teat) number in sheep. A representative example from Chinese Tan sheep, including a normal nipple number (a pair of nipples) and supernumerary nipples [b with three nipples and c four nipples]. Photos were taken by P.K. Figure S6. Location of vertebrae in the sheep skeleton. This includes cervical, thoracic, lumbar, sacral, and coccygeal vertebrae (item source: PNGITEM).

Additional file 3: Supplementary Fig. 3. Upregulated and downregulated signaling pathways and CAZymes in the GEM and WTM. A. Bray-Curtis-based PCoA of the functional pathways. The P-value was based on ANOSIM. The boxplot for the discrete distribution of samples along the PC1 and PC2 axes. B. Significantly changed pathways at level 1 (metabolism pathways) and (C) level 3. The vertical axis represents different pathways, and the horizontal axis represents the proportions of corresponding pathways. (0.01< P ≤0.05 *, 0.001< P ≤0.01 **, P ≤0.001 ***). D. Bray-Curtis-based PCoA of CAZymes. The P-value is based on the ANOSIM. The boxplot shows the discrete distribution of samples along the PC1 and PC2 axes.

Additional file 2: Figure S1. Overlapping or discontinuous peaks in Sanger sequencing of DNA samples obtained from the eight founder animals. Figure S2. Detection of potential off-targeted sites in the eight founder animals by Sanger sequencing. Ten potential off-targeted sites (OT1-OT10) were predicted by Cas-OFFinder [38]. Description: The file contains the results of Sanger sequencing for on- and off-target loci in gene-edited animals.

Additional file 2: Table S1. Additional genomic investigations on sheep tails [159, 218–226]. Table S2. Sheep tails from a transcriptomic perspective: mRNA profiles [4, 12, 14, 20, 21, 219, 227–241]. Table S3. Sheep tails from a transcriptomic perspective: microRNA profiles [242–254]. Table S4. Sheep tails from a transcriptomic perspective: lncRNA profiles [255–259]. Table S5. Sheep tails from a proteomic perspective [260–262]. Table S6. Gene expression and association studies of sheep tails [241, 263–294]. Table S7. Additional genetic investigations on sheep horn phenotype [295–299]. Table S8. Additional genetic studies on sheep coat color phenotype: The ASIP gene [300–306]. Table S9. Additional genetic studies on sheep coat color phenotype: The MC1R gene [89, 302, 304–309]. Table S10. Additional genetic studies on sheep coat color variation [310–327]. Table S11. Additional studies on the curly fleece phenotype in sheep [132, 146, 328–346]. Table S12. Additional studies of the fine vs. coarse fleece phenotype in sheep [138, 333, 334, 347–358]. Table S13. Potential genes linked to the occurrence of the number of thoracic and lumbar vertebrae in sheep [172, 359–362]. Table S14.Credits and courtesies of photos used in the current review article.