CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines

Abstract

Finfish production has seen over three-fold increase in the past 30 years (1990–2020), and Atlantic salmon (A. salmon; salmo salar) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one of the most profitable farmed fish species globally. This growth in production is, however, threatened by a number of problems which can be solved using the CRISPR/Cas technology. In vitro applications of CRISPR/Cas using cell lines can complement its in vivo applications, but salmonids-derived cell lines are difficult to gene edit because they grow slowly, are difficult to transfect and isolate single clones of gene-edited cells. While clonal isolation of the gene-edited Chinook salmon cell line (CHSE-214) has successfully been performed, there is no report of successful clonal isolation of the gene-edited A. salmon ASK-1 and SHK-1cell lines. In the current study, two gene loci—cr2 and mmp9 of A. salmon—were efficiently edited using the ribonucleoprotein (RNP) and plasmid CRISPR/Cas9 strategies. Edited cells were enriched using flow cytometer-activated cell sorting (FACS), followed by clonal isolation and expansion of edited cells. The study both confirms the recent report of the highly efficient editing of these widely used model cell lines, as well as extends the frontline in the single-cell cloning of gene-edited salmonids cells. The report also highlights the pitfalls and future directions in the application of CRISPR/Cas9 in these cells.

Optimising commercial traits through gene editing in aquaculture: Strategies for accelerating genetic improvement

“…402 More recently, Strømsnes et al used a combination of strategies involving both RNP and plasmid CRISPR/Cas9 to edit gene loci in salmonid (Salmo salar) cell lines. 403 The use of RNP complexes for transfection continues to receive much attention for editing within cell lines. 404…”

“…Zoppo et al also utilised an RNP complex and achieved a 39% gene editing efficiency within rainbow trout ( Oncorhynchus mykiss ) cell lines 402 . More recently, Strømsnes et al used a combination of strategies involving both RNP and plasmid CRISPR/Cas9 to edit gene loci in salmonid ( Salmo salar ) cell lines 403 . The use of RNP complexes for transfection continues to receive much attention for editing within cell lines 404 …”

Effect of CRISPR/Cas9 Targets Associated with Iron Metabolism and Its Variation on Transcriptional Regulation of SHK-1 Cell Line as a Model for Iron Metabolism

“…Despite our demonstrated knock-out effect in cells and the use of a lipofectamine reagent as a delivery method, editing efficiency was quite low (1-10%) [6]. However, recent studies have improved genome editing efficiency by over 60% in fish cells using the ribonucleoprotein (RNP) CRISPR-Cas9 strategy [4,7]. This strategy uses electroporation by applying an external electric field.…”

“…In this sense, the use of cell lines derived from fish is a promising tool for studying many key issues of aquaculture. Easier maintenance in flexible temperature regimes and hypoxic conditions makes them preferable in vitro tools over mammalian cell lines [3,4]. Cell lines represent a simplified and more straightforward model to study the potential impact of gene knockouts prior to in vivo trials.…”

Exploring the use of alternative promoters for enhanced transgene and sgRNA expression in Atlantic salmon cells

“…Additionally, in vitro genetic manipulation is a powerful tool to identify the genes underlying desired traits, but tools for transgene expression are often developed in well-studied, distantly related species like human, mouse and zebra sh (Gervais et al, 2021;Pavelin et al, 2021). Although gene editing with plasmids has been accomplished in primary liver cells and SHK-1 cell line of Atlantic salmon (Datsomor et al, 2023;Strømsnes et al, 2022), there are to our knowledge no systematic comparison of endogenous and exogenous promoters in Atlantic salmon. In this study, we aimed to evaluate the activity of Polymerase type III (Pol III) promoters, which are commonly used for expression of short non-coding RNA, and Polymerase type II (Pol II) promoters, which are essential for transgene expression.…”