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

CRISPR‑Cas9 mediated knockout of SYNCRIP impairs PPV

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PPV.NS

Figure 4 Knockout of SYNCRIP inhibits the viral replication in PPV‑infected cells. A

CRISPR‑Cas9 mediated knockout of SYNCRIP impairs PPV
replication
To determine the roles of SYNCRIP in the process of
PPV infection, we used CRISPR/Cas9 genomic edit-
ing system to construct PK-15 cells with SYNCRIP gene
deletion. Two RNA guides (gRNA-63 and gRNA-210)
were designed to target two sites of the exon 1 and exon 2
of SYNCRIP genome sequences respectively (Figure
4
A).
Single cell clones SYNCRIP (63) and SYNCRIP (210)
were selected from cells infected with Cas9 recombinant
lentivirus encoding gRNA-63 or gRNA-210 respectively.
Western blotting analysis shows that SYNCRIP expres-
sion was deficient in the SYNCRIP (210) cells clone, but
was not affected in the SYNCRIP (63) cell clone (Fig-
ure
4
B). We named these two cells as PK
SYNCRIP−/−
and
PK
SYNCRIP+/+
cells, respectively. The cell viability assay
Figure 4 Knockout of SYNCRIP inhibits the viral replication in PPV‑infected cells. A Schematic chromatogram representation of sgRNA
targeting at the SYNCRIP genomic region. PAM sequences are underlined and highlighted in green. sgRNA targeting sites are underlined and
highlighted in red. Red arrows indicate gRNA targeting sites. B Western blotting analysis of the SYNCRIP expression in PK-15 cells infected with
CRISPR/Cas9 lentivirus and then selected by puromycin. The lentiviruses contain gRNA-63 and gRNA-210. C Cell viability of cell lines stably knockout
for SYNCRIP. D‑E Knockout of SYNCRIP inhibits PPV progeny virion production. PK-15, PK
SYNCRIP+/+
and PK
SYNCRIP−/−
cells were infected with PPV
(MOI = 1) for 24 h. The relative fold-change PPV DNA copies and viral titers were determined by q-PCR and TCID
50
assay. The results are shown as the
mean ± SD (n = 3). *p < 0.05 versus PK-15 cells with the same treatment.

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Chen et al. Vet Res (2021) 52:73
shows that the viability of PK
SYNCRIP+/+
and PK
SYNCRIP−/−
cells were not different from PK-15 cells (wild-type)
(Figure
4
C). Next, PK-15, PK
SYNCRIP+/+
and PK
SYNCRIP−/−
cells were infected with equal amounts of PPV (MOI = 1)
for 24 hpi. PPV DNA copies and viral titers significantly
decreased in PK
SYNCRIP−/−
cells compared with PK-15
and PK
SYNCRIP+/+
at 24 hpi (Figures
4
D, E). These results
suggest that SYNCRIP deficiency significantly impaired
PPV replication.

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