Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs
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- 作者：Kristin M. Whitworth ; Joshua A. Benne ; Lee D. Spate…
- 关键词：CRISPR/Cas9 ; Zygote injection ; DNA editing ; gBlock
- 刊名：Transgenic Research
- 出版年：2017
- 出版时间：February 2017
- 年：2017
- 卷：26
- 期：1
- 页码：97-107
- 全文大小：
- 刊物类别：Biomedical and Life Sciences
- 刊物主题：Animal Genetics and Genomics; Plant Genetics & Genomics; Transgenics; Biomedical Engineering/Biotechnology; Genetic Engineering; Molecular Medicine;
- 出版者：Springer International Publishing
- ISSN：1573-9368
- 卷排序：26
文摘
The CRISPR/Cas9 genome editing tool has increased the efficiency of creating genetically modified pigs for use as biomedical or agricultural models. The objectives were to determine if DNA editing resulted in a delay in development to the blastocyst stage or in a skewing of the sex ratio. Six DNA templates (gBlocks) that were designed to express guide RNAs that target the transmembrane protease, serine S1, member 2 (TMPRSS2) gene were in vitro transcribed. Pairs of CRISPR guide RNAs that flanked the start codon and polyadenylated Cas9 were co-injected into the cytoplasm of zygotes and cultured in vitro to the blastocyst stage. Blastocysts were collected as they formed on days 5, 6 or 7. PCR was performed to determine genotype and sex of each embryo. Separately, embryos were surgically transferred into recipient gilts on day 4 of estrus. The rate of blastocyst development was not significantly different between CRISPR injection embryos or the non-injected controls at day 5, 6 or 7 (p = 0.36, 0.09, 0.63, respectively). Injection of three CRISPR sets of guides resulted in a detectable INDEL in 92–100 % of the embryos analyzed. There was not a difference in the number of edits or sex ratio of male to female embryos when compared between days 5, 6 and 7 to the controls (p > 0.22, >0.85). There were 12 resulting piglets and all 12 had biallelic edits of TMRPSS2. Zygote injection with CRISPR/Cas9 continues to be a highly efficient tool to genetically modify pig embryos.