Correction of a Disease Mutation using CRISPR/Cas9-assisted Genome Editing in Japanese Black Cattle

Mitsumi Ikeda, Shuichi Matsuyama, Satoshi Akagi, Katsuhiro Ohkoshi, Sho Nakamura, Shiori Minabe, Koji Kimura, Misa Hosoe

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Isoleucyl-tRNA synthetase (IARS) syndrome is a recessive disease of Japanese Black cattle caused by a single nucleotide substitution. To repair the mutated IARS gene, we designed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create a double-strand break near the mutation site. CRISPR/Cas9 and donor DNA that contained a synonymous codon for the correct amino acid and an Aequorea coerulescens Green Fluorescent Protein (AcGFP) cassette with a piggyBac transposase recognition site at both ends were introduced into bovine fetal fibroblast (BFF) cells isolated from a homozygous mutant calf. Recombinant cells were enriched on the basis of expression of AcGFP, and two cell lines that contained the repaired allele were subcloned. We generated somatic cell nuclear transfer (SCNT) embryos from the repaired cells and transferred 22 blastocysts to recipient cows. In total, five viable fetuses were retrieved at Days 34 and 36. PiggyBac transposase mRNA was introduced into BFF cells isolated from cloned foetuses and AcGFP-negative cells were used for second round of cloning. We transferred nine SCNT embryos to recipient cows and retrieved two fetuses at Day 34. Fetal genomic DNA analysis showed correct repair of the IARS mutation without any additional DNA footprint.

Original languageEnglish
Article number17827
JournalScientific reports
Volume7
Issue number1
DOIs
Publication statusPublished - Dec 1 2017

ASJC Scopus subject areas

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    Ikeda, M., Matsuyama, S., Akagi, S., Ohkoshi, K., Nakamura, S., Minabe, S., Kimura, K., & Hosoe, M. (2017). Correction of a Disease Mutation using CRISPR/Cas9-assisted Genome Editing in Japanese Black Cattle. Scientific reports, 7(1), [17827]. https://doi.org/10.1038/s41598-017-17968-w