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Animal Bioresource in Japan

.: Home > Animal Bioresource in Japan > 2015 > Volume 64 Number 1 > Arata HONDA, Michiko HIROSE, Tadashi SANKAI, Lubna YASMIN, Kazuaki YUZAWA, Kimiko HONSHO, Haruna IZU, Atsushi IGUCHI, Masahito IKAWA, Atsuo OGURA

Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9

Arata HONDA, Michiko HIROSE, Tadashi SANKAI, Lubna YASMIN, Kazuaki YUZAWA, Kimiko HONSHO, Haruna IZU, Atsushi IGUCHI, Masahito IKAWA, Atsuo OGURA
Organization for Promotion of Tenure Track, University of Miyazaki, 5200, Kibara, Kiyotake, Miyazaki 889-1692, Japan
Abstract :

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.

Keywords :
CRISPR/Cas9, gene targeting, rabbit, tyrosinase

Date Deposited : 08 Apr 2015 09:37

Last Modified : 08 Apr 2015 09:37

Official URL: http://https://www.jstage.jst.go.jp/browse/expanim/62/1/_contents

Volume 64, Number 1, - 2015

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