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. 2015 Feb;25(2):258-61.
doi: 10.1038/cr.2014.158. Epub 2014 Nov 28.

One-step generation of p53 gene biallelic mutant Cynomolgus monkey via the CRISPR/Cas system

Haifeng Wan  1 , Chunjing Feng  2 , Fei Teng  2 , Shihua Yang  3 , Baoyang Hu  1 , Yuyu Niu  4 , Andy Peng Xiang  5 , Weizhen Fang  6 , Weizhi Ji  4 , Wei Li  1 , Xiaoyang Zhao  1 , Qi Zhou  1
Affiliations

One-step generation of p53 gene biallelic mutant Cynomolgus monkey via the CRISPR/Cas system

Haifeng Wan et al. Cell Res. 2015 Feb.
No abstract available

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Figures

Figure 1
Figure 1
Generation of p53 gene biallelic mutant monkeys via CRISPR/Cas system. (A) Effects of injection concentrations of Cas9 mRNA/sgRNA on mutagenesis efficiency in monkey embryos. (B) Photograph of monkeys carrying biallelic mutation (#3, left panel) and monoallelic mutation (#1, right panel). (C) Summary of manipulated monkeys embryos and p53 gene mutant monkeys. (D) T7EI assay of the PCR products encompassing the p53 targeting site amplified from the genomic DNA prepared from gonad and umbilical cord of miscarried fetus #m1. Umb, Umbilical cord; Gnd, Gonad. (E) Sanger sequencing of PCR products encompassing the targeting site amplified from the gonad of miscarried fetus #m1. (F) Two rounds of T7EI assay of all the eight tissue samples dissected from live monkey #3. In the first round of T7EI assay, PCR products were re-annealed by themselves, then digested by T7EI, and no indel was detected. In the 2nd round of T7EI assay, the PCR products were annealed with PCR products from wild-type monkeys and then digested. M, Marker; Tal, Tail; Umb, Umbilical cord; Plt, Placenta; Epi, Oral epthelium; Msc, Muscle; Bld, Blood; Skn, Skin; WT, Wild-type. (G) Representative results of Sanger sequencing of PCR products from dissected tissues of #3. The fractions indicate the mutant reads number (numerator) out of total reads number (denominator). (H) Allele-specific PCR reveals that no wild-type allele can be detected in the eight dissected tissues of monkey #3. (I) Allele-specific RT-PCR reveals that no wild-type p53 mRNA can be detected in monkey #3. (J) Sequences from HDR-modified embryos showing correct integration of the TAA stop codon into p53 locus. (K) Sanger sequencing of PCR products encompassing the HDR-editing site from precisely modified embryos. The PAM sequence is shown in red bold uppercase. Targeted integration (KI) and the sizes of insertion (+), deletion (Δ) are presented on the right of each allele.
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References

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