Volume 156, Issue 4, Pages 836-843 (February 2014) Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos  Yuyu Niu, Bin Shen, Yiqiang Cui, Yongchang Chen, Jianying Wang, Lei Wang, Yu Kang, Xiaoyang Zhao, Wei Si, Wei Li, Andy Peng Xiang, Jiankui Zhou, Xuejiang Guo, Ye Bi, Chenyang Si, Bian Hu, Guoying Dong, Hong Wang, Zuomin Zhou, Tianqing Li, Tao Tan, Xiuqiong Pu, Fang Wang, Shaohui Ji, Qi Zhou, Xingxu Huang, Weizhi Ji, Jiahao Sha  Cell  Volume 156, Issue 4, Pages 836-843 (February 2014) DOI: 10.1016/j.cell.2014.01.027 Copyright © 2014 Elsevier Inc. Terms and Conditions

Cell 2014 156, 836-843DOI: (10.1016/j.cell.2014.01.027) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 sgRNA:Cas9-Mediated Modifications of Nr0b1, Ppar-γ, and Rag1 in COS-7 Cells (A) Schematic diagram of sgRNAs targeting at Nr0b1, Ppar-γ, and Rag1 loci. PAM sequences are underlined and highlighted in green. sgRNA targeting sites are highlighted in red. (B) Detection of sgRNA1:Cas9-mediated cleavage of Nr0b1, Ppar-γ, and Rag1 by PCR and T7EN1 cleavage assay. M, DNA marker; sg1, sgRNA1; sg2, sgRNA2; Con, control. (C) Sequences of modified Nr0b1, Ppar-γ, and Rag1 loci detected in COS-7 cells. At least 15 TA clones of the PCR products were analyzed by DNA sequencing. The PAM sequences are underlined and highlighted in green; the targeting sequences in red; the mutations in blue, lower case; deletions (−), insertions (+). N/N indicates positive colonies out of total sequenced. Cell 2014 156, 836-843DOI: (10.1016/j.cell.2014.01.027) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 sgRNA:Cas9-Mediated Modifications of Nr0b1, Ppar-γ, and Rag1 in Cultured Embryos (A) Detection of sgRNA1:Cas9-mediated on-target cleavage of Nr0b1, Ppar-γ, and Rag1 by T7EN1 cleavage assay. PCR products were amplified and subjected to T7EN1 cleavage assay. Samples with cleavage bands were marked with an asterisk “∗.” (B) DNA sequences of marked samples. TA clones from the PCR products were analyzed by DNA sequencing. Mutations in three PCR products (labeled with red asterisk) indentified by T7EN1 cleavage assay were not detected by TA sequencing because of limited amount of colonies. The PAM sequences are underlined and highlighted in green; the targeting sequences in red; the mutations in blue, lower case; deletions (−), and insertions (+). N/N indicates positive colonies out of total sequenced. See also Figure S1. Cell 2014 156, 836-843DOI: (10.1016/j.cell.2014.01.027) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 sgRNA:Cas9-Mediated Modifications of Ppar-γ and Rag1 in Founder Cynomolgus Monkeys (A) Photographs of 14-day-old founder infants A and B. (B) PCR products of the target region of Ppar-γ and Rag1 in founders. Targeted region of Ppar-γ and Rag1 loci were PCR amplified from the umbilical cord genomic DNA of A and B founders. M, DNA marker; Con, control umbilial cord from wild-type cynomolgus monkey, which was born 9 days after birth of A and B. (C) Detection of sgRNA:Cas9-mediated on-target cleavage of Ppar-γ and Rag1 by T7EN1 cleavage assay. PCR products from (B) were subjected to T7EN1 cleavage assay. (D) Sequences of modified Ppar-γ and Rag1 loci detected in founders. At least 18 TA clones of the PCR products were analyzed by DNA sequencing. The PAM sequences are underlined and highlighted in green; the targeting sequences in red; the mutations in blue, lower case; deletions (−), and insertions (+). N/N indicates positive colonies out of total sequenced. See also Figure S2 and S4. Cell 2014 156, 836-843DOI: (10.1016/j.cell.2014.01.027) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 sgRNA:Cas9-Mediated Modifications of Nr0b1, Ppar-γ, and Rag1 in Ear and Placenta of Founders (A) PCR products of the targeted region of Nr0b1, Ppar-γ, and Rag1 in founders. Target regions of Nr0b1, Ppar-γ, and Rag1 loci were PCR amplified from the ear and placenta genomic DNA of A and B founders. M, DNA marker; Con, wild-type control. (B) Detection of sgRNA1:Cas9-mediated on-target cleavage of Nr0b1, Ppar-γ, and Rag1 by T7EN1 cleavage assay. (C) DNA sequences of Nr0b1, Ppar-γ, and Rag1 loci. The PCR products were analyzed by DNA sequencing. The PAM sequence are underlined and highlighted in green; the targeting sequences in red; the mutations in blue, lower case; deletions (−), and insertions (+). N/N indicates positive colonies out of total sequenced. See also Figure S3 and S4. Cell 2014 156, 836-843DOI: (10.1016/j.cell.2014.01.027) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S1 sgRNA:Cas9-Mediated Modifications of Nr0b1, Ppar-γ and Rag1 in Cultured Embryos, Related to Figure 2 PCR products of the targeted region of Nr0b1, Ppar-γ, and Rag1 were amplified from cultured embryos. M, DNA marker. Con, wild-type control blastocyst without injection. Cell 2014 156, 836-843DOI: (10.1016/j.cell.2014.01.027) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S2 sgRNA:Cas9-Mediated Modifications of Nr0b1 in Founder Cynomolgus Monkeys, Related to Figure 3 sgRNA1:Cas9-mediated cleavage of Nr0b1 in founders A and B were detected by PCR and T7EN1 cleavage assay using umbilical cord genomic DNA as template. M, DNA marker. Con, wild-type control. Cell 2014 156, 836-843DOI: (10.1016/j.cell.2014.01.027) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S3 Derivation of Targeted Allele Analysis by SNP Mapping, Related to Figure 4 (A) Schematic diagram of location of the single-nucleotide polymorphism (SNP) downstream of Rag1 sgRNA target site in founder twins’ parents. The Rag1 allele was amplified by primers 3.8F and 3.8R using the ear genomic DNA of founder twins’ parents as template. PCR products were sequenced by indicated primers. Four different SNPs were detected (purple). See also Table S1. (B) Genotype of founder twins’ parents was determined by TA cloning and sequencing of PCR products from (A). Father’s alleles were highlighted in red, mother’s in green. (C) Founder twins’ genotypes were determined by TA cloning and sequencing of PCR products amplified by primers 3.8F and 3.8R. (D) In founder B, mutations were detected in alleles from both parents. N/N indicates positive colonies out of total sequenced. Cell 2014 156, 836-843DOI: (10.1016/j.cell.2014.01.027) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S4 sgRNA:Cas9-Mediated Modifications of Ppar-γ in Founder Twins’ Parents and Surrogate Mother, Related to Figures 3 and 4 (A) Detection of sgRNA:Cas9-mediated on-target cleavage of Ppar-γ by T7EN1 cleavage assay. PCR products of the target region of Ppar-γ amplified from the ear genomic DNA of parents and surrogate mother were subjected to T7EN1 cleavage assay. No typical cleavage band was detected. Fa, father; Mo, mother; SM, surrogate mother. (B) Sequences of Ppar-γ locus in parents and surrogate mother. At least 10 TA clones of the PCR products were analyzed by DNA sequencing. N/N indicates positive colonies out of total sequenced. Cell 2014 156, 836-843DOI: (10.1016/j.cell.2014.01.027) Copyright © 2014 Elsevier Inc. Terms and Conditions