Additional file 1 of Generation of sheep with defined FecBB and TBXT mutations and porcine blastocysts with KCNJ5G151R/+ mutation using prime editing
Creators: name: Zhou, Shiwei
name: Lenk, Laura Johanna
name: Gao, Yawei
name: Wang, Yuhui
name: Zhao, Xiaoe
name: Pan, Menghao
name: Huang, Shuhong
name: Sun, Kexin
name: Kalds, Peter
name: Luo, Qi
name: Lillico, Simon
name: Sonstegard, Tad
name: Scholl, Ute I.
name: Ma, Baohua
name: Petersen, Bjoern
name: Chen, Yulin
name: Wang, Xiaolong
Publisher: name: figshare
Year: 2023
Subjects: subject: Space Science
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: Evolutionary Biology
subject: Biological Sciences not elsewhere classified
subject: Mathematical Sciences not elsewhere classified
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology

description: 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.