Cell cultures
CRFK (CCL-94, ATCC, Manassas, VA, USA), AH927 (feline embryonic fibroblasts)36, and a feline sarcoma-positive leukemia-negative (S+L−) fibroblast cell line termed QN10S cells37 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Equine-biotech, Bengaluru, India), penicillin (100 IU/mL), and streptomycin (100 ng/mL) (Invitrogen, MA, USA ). FL74 cells (feline lymphoblasts) (ATCC CRL-8012)38 were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated FCS, penicillin (100 IU/mL), and streptomycin (100 ng/mL).
Construction of TALEN plasmids
A two-step Golden Gate assembly method with the Platinum Gate TALEN Kit (Addgene, MA, USA)30 was used to construct Platinum TALEN plasmids containing the homodimer-type FokI nuclease domain. TALENs were designed against the RDRS env gene. Target sequences for TALENs are summarized in Fig. 1.
Knockout of RDRS env in CRFK cells
CRFK cells (5 × 105) were suspended in 100 µL of R buffer (Neon Transfection system, Invitrogen) and then electroporated with 1.8 µg of each TALEN plasmid and 0.36 µg of a puromycin-resistant gene expression plasmid to select TALEN-transfected cells under the following conditions: pulse voltage, 1400 V; pulse width, 20 ms; and pulse number, 2. This transfection procedure was repeated twice.
Cel-I assay and sequencing
Two days after transfection, genomic DNA was extracted from CRFK cells using QIAamp DNA Blood Mini kit (QIAGEN, CA, USA). PCR was performed using PrimeSTAR GXL polymerase (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. Primers used for this PCR were listed in Table 2. The amplicons were heat-denatured, digested by Surveyor nuclease (Transgenomic, NE, USA), and subjected to agarose gel electrophoresis to detect TALEN-induced mutations.
The PCR products were also cloned into pCR BluntII TOPO plasmid vector (Life Technologies, CA, USA) and sequenced. Sequencing was performed by a commercial DNA sequencing service (FASMAC, Kanagawa, Japan). Alignment of nucleotide sequences and estimation of homology were performed using GENETYX win Ver.11 (GENETYX, Tokyo, Japan).
Selection of TALEN-transfected CRFK cells
TALEN-transfected CRFK cells were seeded in 10-cm dishes and selected with 4 μg/mL of puromycin (InvivoGen, CA, USA). Then, AH927 cells, which do not contain RDRS A2 or C1 in their genomes (thus, AH927 cells are RD-114 non-producer cells), were co-cultured as feeder cells to supply the nutrients for the small colonies. Medium with puromycin was replaced every 3 days. One month after starting selection with puromycin, individual puromycin-resistant cell colonies were picked up and cultured in 96-multiwell plates. Two days after transferring the cells into 96-multiwell plates, the cells were further subcultured in 6-multiwell plates. The clones were then subjected to PCR and sequencing analyses using genomic DNA.
Quantification of RD-114 viral production by real-time RT-PCR
The untreated parental CRFK cells and cloned RDRS env knockout CRFK cells (RDKO_CRFK cells) were cultured for 3 days. Culture supernatants were collected, filtrated through 0.45-µm filters, and then pretreated with DNase I (Roche Diagnostics, IN, USA). The copy number of RDRS env RNA in the culture supernatants was measured by real-time RT-PCR. Real-time RT-PCR was performed using TaKaRa Ex Taq HS DNA polymerase (TaKaRa) according to the manufacturer’s instructions. A TaqMan probe (Applied Biosystems, MA, USA) and primers used for real-time RT-PCR were listed in Table 2.
Titration of infectious RD-114 virus
Replication-competent gammaretrovirus in the culture supernatants of parental CRFK and RDKO_CRFK cells was evaluated by the S+L− assay using QN10S cells39. QN10S cells were seeded in 24-well plates at 1 × 104 cells/well one day before infection and diluted supernatants of RDKO_CRFK cells were used to inoculate QN10S cells in the presence of polybrene (Sigma-Aldrich) (8 µg/mL). Seven days after inoculation, numbers of foci were counted.
Cell proliferation assay
Cell proliferations of parental CRFK and RDKO_CRFK cells were measured using Cell Proliferation Kit I (MTT) (Roche Diagnostics). The cells were seeded at 1 × 105 cells/well in 96-multiwell plates and then cultured for 2 days. The assay was performed according to the manufacturer’s instructions. The spectrophotometric absorbance of the sample was measured at a wavelength of 595 nm using a Wallac 1420 ARVOsx (PerkinElmer Life Sciences, MA, USA).
Infection and titration of feline herpesvirus type 1, calicivirus, and panleukopenia virus
FHV-1 strain 00-01540, FCV strain 01-106, and FPLV strain V14241 were used. To evaluate the growth kinetics of FHV-1 strain 00-015, 1000-fold dilutions of a virus stock are prepared, and 60 µL aliquots are used to inoculate parental CRFK and RDKO_CRFK cell monolayers in 6-multiwell plates. The cells were inoculated with FCV strain 01-106 at a multiplicity of infection (MOI) of 0.01 and FPLV strain V142 at MOI of 0.1. After incubation at 37 °C for 1 h, the cells were washed three times with PBS and then cultured with 2 mL of DMEM supplemented with 10% FCS for 4, 2, and 6 days, respectively. The titers of FHV-1 and FCV produced in parental CRFK or RDKO_CRFK cells were measured in TCID50 using CRFK cells, as described previously40,42. The titers of FPLV produced in parental CRFK or RDKO_CRFK cells were measured in TCID50 using FL74 cells, as described previously43,44.
Off-target analysis
The Paired Target Finder (https://tale-nt.cac.cornell.edu/)45 was used to identify potential off-target sites for RDRS env. Score cutoff and spacer lengths were set to 3.0 and 10–30, respectively. Genomic regions around each candidate site were amplified by PCR using primers listed in Table 2 and the sequences were confirmed by direct sequencing.
