CRISPR/Cas9 as tool for functional study of genes involved in preimplantation embryo development

PloS One
Jeongwoo KwonNam-Hyung Kim

Abstract

The CRISPR/Cas9 system has proven to be an efficient gene-editing tool for genome modification of cells and organisms. However, the applicability and efficiency of this system in pig embryos have not been studied in depth. Here, we aimed to remove porcine OCT4 function as a model case using the CRISPR/Cas9 system. Injection of Cas9 and single-guide RNA (sgRNA) against OCT4 decreased the percentages of OCT4-positive embryos to 37-50% of total embryos, while ~100% of control embryos exhibited clear OCT4 immunostaining. We assessed the mutation status near the guide sequence using polymerase chain reaction (PCR) and DNA sequencing, and a portion of blastocysts (20% in exon 2 and 50% in exon 5) had insertions/deletions near protospacer-adjacent motifs (PAMs). Different target sites had frequent deletions, but different concentrations of sgRNA made no impact. OCT4 mRNA levels dramatically decreased at the 8-cell stage, and they were barely detectable in blastocysts, while mRNA levels of other genes, including NANOG, and CDX2 were not affected. In addition, the combination of two sgRNAs led to large-scale deletion (about 1.8 kb) in the same chromosome. Next, we injected an enhanced green fluorescent protein (eGFP) vector targeting th...Continue Reading

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Citations

May 19, 2018·Scientific Reports·Bradford W DaigneaultPablo J Ross
May 24, 2016·The Journal of Reproduction and Development·Natsuko EmuraKen Sawai
Nov 27, 2020·Molecular Reproduction and Development·Wei GaoXianfeng Yu
Jun 22, 2020·Journal of Equine Veterinary Science·Sergio Navarro-SernaPablo Juan Ross

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Methods Mentioned

BETA
antisense oligonucleotide
PCR
in vitro transcription
genetic knockout
antisense oligonucleotides

Software Mentioned

R R
TALEN

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