Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes

Abstract

Infectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide valuable models to study infectious diseases in salmonids, and genome editing using CRISPR/Cas systems provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas editing has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly used salmonid fish cell lines: Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90% cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture.

The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon

“…Firstly, CRISPR-Cas9 genome editing was used to KO the nae1 gene in SHK-1 cells using recombinant Cas9 protein and custom synthesised gRNAs; a method for high specificity editing of target genes in salmonid cell cultures [ 26 ]. Exon 2 of the Atlantic salmon nae1 locus was targeted and editing efficiency was 93–97% resulting in 82–87% frameshift mutation (depending on the replicate), highlighting that the vast majority of cells in the mixed cell population were successfully edited.…”

“…CRISPR-Cas9 gRNAs were designed for nae1 and cdh1 and selected for maximum on-target efficiency, and minimum off-targets, using the benchling ( www.benchling.com ) and the Synthego CRISPR design tools. Nae1 KO and cdh1 KO SHK-1 cells were produced by using method described in [ 26 ]. Briefly, SHK-1 cells were transfected with 1 μM Cas9 ribonucleoprotein targeting exon 2 of nae1 or cdh1 ( Supplementary Table 1 ) by electroporation with 2 pulses at 1400 V for 20 ms. Genomic DNA was extracted at 7 days post electroporation, the target region was amplified by PCR ( Supplementary Table 1 ), and gene-editing efficiency was assessed by Sanger sequencing and ICE analysis ( https://ice.synthego.com ), showing 94 and 93% editing efficiency in nae1 KO and cdh1 KO SHK-1 cells, respectively.…”

Genome editing and its applications in genetic improvement in aquaculture

“…52 For example, in the pSpCas9(BB)-2A-gfp vector system or Lentivirus CRISPR system, gRNA sequence was cloned into the vector and then was transfected into cells for gene knockout. [53][54][55] For in vitro gene editing in cells, a vector and/or lentivirus system still have to be used. 56 Alternatively, a cloning-free approach was developed.…”

“…Figure 3). Other methods to deliver genome editing components into one-cell fertilised eggs or cultured cell have been developed in fish, such as lentivirusmediated methods, 54 electroporation, 27,69 and ribonucleoprotein complexes. 54 Methods for measuring off-target guide RNA activity have also been developed.…”

“…Other methods to deliver genome editing components into one-cell fertilised eggs or cultured cell have been developed in fish, such as lentivirusmediated methods, 54 electroporation, 27,69 and ribonucleoprotein complexes. 54 Methods for measuring off-target guide RNA activity have also been developed. 70 For example, to quantify unintended off-target DNA double-strand breaks on a whole genome in cultured cells transfected with CRISPR enzymes, a detailed protocol based on the breaks labelling in situ and sequencing method was developed.…”