Syncrip facilitates porcine parvovirus viral dna replication through the alternative splicing of ns1 mrna to promote ns2 mrna formation

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Chuyển đổi dữ liệu	07.01.2023
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PPV.NS

CRISPR‑Cas9 mediated SYNCRIP knockout in PK‑15 cells

Antibodies and reagents
Antibodies include monoclonal mouse anti-SYNCRIP
antibody (ab10687; abcam, Cambridge, MA, UK),
monoclonal mouse anti-capsid primary antibody 3C9
(3C9-D11-H11; ATCC, Manassas, VA, USA), β-actin
(13C000500; GenScript, Nanjing, China), Normal mouse
IgG antibody (A7028; Beyotime, Shanghai, China), HRP-
conjugated anti-mouse IgG (RK244131; Invitrogen,
Waltham, MA, USA). T7-Biotin Labeling Transcrip-
tion Kit (R11074.1; Ribo, Guangzhou, China), Dyna-
beads
™
MyOne
™
streptavidin C1 (65002; ThermoFisher,
Waltham, MA, USA), Protein A PLUS-Agarose and Pro-
tein G PLUS-Agarose (SC-2002; Santa, Beijing, China).
CRISPR‑Cas9 mediated SYNCRIP knockout in PK‑15 cells
Targeting sites in the SYNCRIP gene were selected using
the CRISPR program (Genome Engineering, Broad Insti-
tute Cambridge, MA, USA). CRISPR-Cas9 designing and
analysis methods were described in a previous study [
30
,
31
]. In brief, two pairs of gRNA specifically targeting the
porcine SYNCRIP sequence were designed using the
optimized CRISPR design tool. Oligonucleotides were
annealed and ligated into the Lenti-CRISPRv2 plasmid
(Addgene, #52961) using the BsmB I sites, respectively,
and confirmed by sequencing analysis. HEK293T cells
were transfected with recombinant plasmids accom-
panied with psPAX2 (Addgene, #12260) and pMD2.G

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Chen et al. Vet Res (2021) 52:73
(Addgene, #12259) to obtain recombinant lentiviruses
at 72 h. Then, the culture supernatant was collected
and used to infect PK-15 cells. After 48 h, the cells were
selected by puromycin at a concentration of 10 μg/mL.
Positive cells were obtained after about 2 weeks and then
subcloned into 96-well plates for single-clone growth and
saved as cell stocks.

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