Cell Lines
The cell lines used in this study were as follows: (i) salmon head kidney 1 (SHK-1), an immortalised cell line from Atlantic salmon (Salmo salar) obtained from the European Collection of Authenticated Cell Cultures (ECACC) (97111106); (ii) Atlantic salmon kidney (ASK), an immortalised cell line from Atlantic salmon (S. salar) obtained from American Type Culture Collection (ATCC; CRL-2747); (iii) rainbow trout gonad (RTG-2), an immortalised cell line from rainbow trout (O. mykiss) obtained from ECACC (90102529); and (iv) Chinook salmon embryo 214 (CHSE-214), an immortalised cell line from Chinook salmon (O. tshawytscha) obtained from ECACC (91041114). All cells were grown as a monolayer in L15 media (Sigma-Aldrich, St. Louis, USA) supplemented with heat-inactivated foetal bovine serum (FBS) (Gibco, Waltham, USA) (SHK-1, 5%; RTG-2, CHSE-214 and ASK, 10%), 40 μM β-mercaptoethanol (Gibco) for SHK-1, 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco). All cells were cultured in an incubator at 22 ± 1 °C without CO2. SHK-1 was split 1:2 at 80% confluency with
conditioned media and the rest of cell lines were split 1:3–1:4 with fresh media.
SHK-1 cells were used for the initial optimisation of RNP editing, as described below. As part of this process, an SHK-1 line with a GFP transgene was created to allow testing of gRNA targeting knockout of this transgene causing loss of fluorescence. This cell line (SHK-FuGFP) was generated by transfecting a CMV-GFP_Puromycin construct (Addgene 45561, a gift from Michael McVoy) in SHK-1 cells with Fugene HD transfection reagent (Promega, Madison, USA). To achieve this, SHK-1 cells were plated in a 24-well plate at 40,000 cells per well and incubated overnight at 22 °C. Media was replaced with 500 μL of L15 (10% FBS, no antibiotics) containing 0.5 μg plasmid and 1.5 μL of FugeneHD (ratio Fugene:DNA 3:1, according to manufacturer’s instructions). After 7 days, cells were selected with puromycin at a concentration of 1 μg/mL for a period of 4 weeks.
Optimisation of Cas9 RNP Transfection and Editing
To test and optimise the Cas9 RNP platform, an intergenic region of the Atlantic salmon genome (GenBank accession NC_027325.1 ssa26; 15004350–15004900) was targeted with a gRNA via transfection of the SHK-1 cell line. This was followed by validation of the optimised conditions by EGFP knockout in the SHK-FuGFP cell line and knockout of coding region of slc45a2 (Gene ID: 106563596).
The crRNAs were designed with CRISPOR (http://crispor.tefor.net/) and the CRISPR Design Tool (Synthego Inc., Menlo Park, USA), and crRNAs and tracrRNAs were ordered from IDT (details of all gRNA are given in Table 1). The RNP complexes were assembled as follows: crRNA and tracrRNA were resuspended in nuclease-free water at 100 μM, aliquoted and frozen at − 80 °C. One microlitre of crRNA and 1 μL of tracrRNA were mixed and incubated at 95 °C for 5 min. The mixture was cooled to room temperature, and 2 μL of 20 μM Cas9 (NEB, Ipswich, USA) was added (final concentration of 10 μM of Cas9 and 25 μM of gRNA). The complexes were incubated at room temperature for 15 min and kept on ice until use.
Table 1 Guide RNA sequences and primers used for amplification and sequencing of target genomic regions The first optimisation step involved varying the concentration of the Cas9-gRNA RNP complex, with the starting point being electroporation conditions that have previously been successful in plasmid transfection of SHK-1 (data not shown). To achieve this, different concentrations of RNP were diluted in OptiMEM reduced serum media (Gibco) (final volume 4 μL) and mixed with 10 μL of SHK-1 cells at 107 cells/mL in OptiMEM (final concentrations range from 0.0875 to 2.8 μM Cas9 RNP). After 5-min incubation at room temperature, the cells plus the RNP were electroporated with the Neon system (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions but with OptiMEM instead of Neon R Buffer (Invitrogen). The mixture was electroporated using 10 μL tips and dispensed in 1 mL of fresh media in a 24-well plate. One hundred microlitres of the suspension was transferred to a 96-well plate (104 cells) for genomic DNA isolation or cell culture. The cells were incubated overnight at room temperature, and the media changed to 1 mL of fresh media. Once the cells reached confluency (in 24-well plates), they were resuspended in media (using trypsin) and divided 1:2 (adding 33% conditioned media for the SHK-1 cells). Cells were kept in 96- or 24-well plates for gDNA isolation at 1, 2, 4, 7, and 14 days post treatment (dpt) or expanded to 6-well plates once they reached confluency for measurement of fluorescence using flow cytometry at 14 dpt.
Cell Survival
In addition to assessing the transfection and editing efficiency of the SHK-1 cells, the cell viability was tested in parallel for each of the setting used for the optimisation protocol using CellTiter-Glo 2.0 (Invitrogen) and cells with Cas9 RNP complex but not electroporated as controls. In brief, following electroporation, 100 μL of the cells in the 24-well plate were transferred to a 96-well plate and incubated for 48 h. Surviving cells still attached to the bottom of the plate were rinsed once in PBS and 120 μL of CellTiter-Glo solution (diluted 1:10 in PBS) was added to each well. The plate was incubated in the dark for 30 min on a plate rocker at room temperature and 100 μL of the solution was transferred to a flat bottom white wall 96-well plate (Greiner Bio-One, Austria). The luminescence was measured using a Cytation3 imaging reader and the Gen5 software V3.03 (BioTek, Winooski, USA).
Validation of Optimised Cas9 RNP Editing by GFP Knockout
A second test of RNP editing was performed in the SHK-fuGFP cells by using the optimised settings to transfect an RNP complex with a gRNA targeting knockout of the GFP transgene. Following the transfection, the loss of GFP was measured by flow cytometry. To achieve this, the cells were trypsinised and resuspended in PBS. The cells were kept on ice and flow cytometry was performed using a Fortessa-X20 (BD Biosciences, San Jose, USA). Single-cell events were gated, and the percentage of GFP-positive cells and the intensity of GFP fluorescence from each cell was measured.
Assessing the Efficiency and Nature of the Edits Using Sanger Sequencing
Testing of editing efficiency was performed by isolation of genomic DNA followed by PCR amplicon Sanger sequencing. Genomic DNA (gDNA) was extracted with QuickExtract buffer (Lucigen, Middleton, USA) by adding 30 μL to a well of a 96-well plate and incubating for 5 min. The samples were then processed according to the manufacturer’s instructions (65 °C for 15 min and 98 °C for 2 min). PCR was performed with 50 μL reactions using NEB Q5 and 1 μL of the gDNA with 33 cycles of amplification at optimal annealing temperature (Table 1). Five microlitres of the PCR product was run on a 1.5% agarose gel to verify correct amplification. Amplified sequence was purified with AmPURE XP magnetic beads (Agencourt, Beverly, USA) according to the manufacturer’s instructions (using 1:1 ratio) and sent to GATC/Eurofins (Germany) for Sanger sequencing. Analysis of the chromatograms (based on .abi files) was used to assess the editing efficiency and nature of the induced edits using the Inference of CRISPR Edits (ICE, Synthego Inc) software for Cas9 or TIDE software (Brinkman et al. 2014) for Cas12a to determine the editing efficiency (% of cells containing putative indels).
Testing of RNP Editing in Other Salmon Cell Lines
Following optimising of the electroporation and incubation settings for the SHK-1 cells described above, similar protocols were tested in the other three cell lines by targeting the slc45a2 gene. Several combinations of different electroporation settings (1200–1600 V, 10–40 ms, 1–3 pulses) and cell resuspension buffers (Neon R buffer and OptiMEM) were tested to achieve highest transfection rate using tracrRNA–ATTO550 (IDT, Coralville, USA) by detecting ATTO550-positive cell population using flow cytometry at 24 h post electroporation. The best transfection result (99.9–100%) was obtained with 1400 V 20 ms 1 pulse for RTG-2 and 1600 V 10 ms 3 pulses for CHSE-214 and ASK with OptiMEM as a resuspension buffer (data not shown).
The Cas9 RNP complex was assembled as described above and Cas12a RNP was formed by adding 31.2 pmol of AsCas12a (IDT) and 50 pmol of crRNA (IDT) per 105 cells. The complexes were incubated at room temperature for 15 min and kept on ice until use. The final concentration of 1 μM of Cas9 RNP and 2.6 μM of AsCas12a RNP were tested with the optimised electroporation settings for each cell line. At 7 dpt, the editing efficiency and the nature of the induced edits were assessed by Sanger sequencing and ICE software for Cas9 or TIDE software for Cas12a as described above.