Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.

Carola E Dehler,Bertrand Collet,Samuel A M Martin,Pierre Boudinot,Pierre Boudinot

doi:10.1007/s10126-016-9708-6

Abstract

CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.

Highlights

The CRISPR/Cas system was initially discovered in the genome of Escherichia coli (Ishino et al 1987), and later found in about 40 % of bacteria, and a great majority of Archae (Horvath and Barrangou 2010)
PCR analysis of cDNA made from RNA purified from the CHSE-EC clonal cell line showed a strong expression of nCas9n when compared to mock cDNA produced by omitting reverse transcriptase
The 192 nucleotide Cas9 fragment could be amplified by PCR directly from genomic DNA purified from CHSEEC, indicating that at least one copy of the plasmid should be integrated in the genome of the CHSE-EC cell line

Summary

Introduction

The CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) system was initially discovered in the genome of Escherichia coli (Ishino et al 1987), and later found in about 40 % of bacteria, and a great majority of Archae (Horvath and Barrangou 2010). The isolation and characterisation of the Cas protein enabled to establish a method for genome editing in a wide range of organisms generating knockout models very efficiently and very quickly using small Bguide RNA molecules^ sgRNA in which cr and tracrRNA are fused (Ran et al 2013; Sander and Joung 2014; Hsu et al 2014). This system has been used in a number of species including fish, to generate lines of sitedirected mutated animals by egg manipulation: zebrafish (Hruscha et al 2013; Hwang et al 2013; Kimura et al 2014; Jao et al 2013; Irion et al 2014), Atlantic salmon This communication presents an efficient method to generate CRISPR/Cas knockout in the Chinook salmon Oncorhynchus tshawytscha cell line, CHSE-214

Methods

Results

Conclusion