Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation.

Molecular Biology Reports
Maki HirataTakeshige Otoi

Abstract

The CRISPR/Cas9 system now allows for unprecedented possibilities of genome editing. However, there are some limitations, including achieving efficient one-step multiple genome targeting to save costs, time, and ensure high quality. In the present study, we investigated the efficiency of one-step multiple gene modification by electroporation in porcine zygotes using pooled guide RNAs (gRNAs) targeting CMAH, GHR, GGTA1, and PDX1. We first selected the best-performing gRNA from three different designs for each gene based on the effect on embryo development and mutation efficiency. The three gRNAs showed equivalent effects on the rates of blastocyst formation in each targeted gene; however, gRNAs CMAH #2, GHR #3, GGTA1 #3, and PDX1 #3 showed the highest biallelic mutation rate, although the total mutation rate of PDX1 #3 was significantly lower than that of PDX1 #1. Therefore, CMAH #2, GHR #3, GGTA1 #3, and PDX1 #1 were used as a mixture in electroporation to further clarify whether multiple genes can be targeted simultaneously. Individual sequencing of 43 blastocysts at the target sites of each gene showed mutations in one and two target genes in twenty-four (55.8%) and nine (20.9%) blastocysts, respectively. No mutation was dete...Continue Reading

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Citations

Sep 27, 2020·In Vitro Cellular & Developmental Biology. Animal·Maki HirataTakeshige Otoi
May 1, 2021·Frontiers in Genetics·Jason C Lin, Alison L Van Eenennaam
Apr 13, 2021·The Journal of Reproduction and Development·Fuminori TaniharaTakeshige Otoi

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Methods Mentioned

BETA
PCR

Software Mentioned

STATVIEW

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