Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish

G3: Genes, Genomes, Genetics

article

Peinan Hu¹  Xueying Zhao¹  Qinghua Zhang¹  Weiming Li¹  Yao Zu¹ 

[1] International Research Center for Marine Biosciences, Ministry of Science and Technology, College of Fisheries and Life Sciences, Shanghai Ocean University, 201306, P.R. China;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, College of Fisheries and Life Sciences, Shanghai Ocean University, 201306, P.R. China;National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, P.R. China;Department of Fisheries and Wildlife, Michigan State University, East Lansing, Michigan 48824

关键词: CRISPR/Cas9;    Cas9 protein;    knockout;    efficiency;    NLS;    zebrafish;   

DOI  :  10.1534/g3.117.300359

学科分类：社会科学、人文和艺术（综合）

来源: Genetics Society of America

【 摘 要 】

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites ( tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system.

【 授权许可】

CC BY|CC BY-NC