Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo

Author:

Zhang Zeming1,Wang Jie12,Li Jianeng1,Liu Xiang1,Liu Lei1,Zhao Changle1,Tao Wenjing1,Wang Deshou1ORCID,Wei Jing1ORCID

Affiliation:

1. Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, China

2. Sichuan Province Yuechi Middle School, Guang’an 638300, China

Abstract

Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a specific site in medaka (Oryzias latipes) in vitro and in vivo. By using the enhanced green fluorescent protein reporter plasmid pGNtsf1, we demonstrate that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) mediated very high gene editing efficiency in medaka cultured cells, but not by exogenous U6 promoters. After optimizing the conditions, the gene editing efficiencies of eight sites targeting for four endogenous genes were calculated, and the highest was up to 94% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA also mediated efficient gene knock-out in vivo. Furthermore, pCas9-mU6sgRNA efficiently mediated gene knock-in at a specific site in medaka cultured cells as well as embryos. Collectively, our study demonstrates that the genetic relationship of U6 promoter is critical to gene editing efficiency in medaka cultured cells, and a simple and efficient system for medaka genome editing in vitro and in vivo has been established. This study provides an insight into other fish genome editing and promotes gene functional analysis.

Funder

National Natural Science Foundation of China

the Chongqing Natural Science Foundation of China

Publisher

MDPI AG

Subject

General Agricultural and Biological Sciences,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology

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