Effects of Knockout of Gene SERPINF2 via CRISPR/cas9 on the Replication of Bovine Viral Diarrhea Virus

doi:10.13560/j.cnki.biotech.bull.1985.2020-0886

Abstract

Abstract:

This work is aimed to knock out the gene SERPINF2 in MDBK cells by CRISPR/cas9 gene editing technology,and to explore the effect of gene SERPINF2 on the replication of bovine viral diarrhea virus（BVDV）. CRISPR/Cas9 technology was performed to establish SERPINF2-knockout cells. Quantitative real-time PCR（qRT-PCR）,immunofluorescence staining and Reed-Muench assay were applied to detect the viral RNA levels,the accumulation of double strand RNA（dsRNA）,viral titer,and cytopathic effect（CPE）in the BVDV infected by SERPINF2-knockout cells.The SERPINF2-knockout MDBK cells SERPINF2 KO were successfully constructed. Of the 27 positive clones,19 clones were sequenced with frame-shift mutations,such as deletion（-）and insertion（+）. The knockout efficiency was approximately 70.37%. The SERPINF2 mRNA transcription level significantly increased after the BVDV TC strain infected MDBK cells. Compared with the scramble cells in the control group,viral RNA levels and dsRNA accumulation decreased significantly in the SERPINF2 KO cells infected with BVDV TC,CPE delay occurred,and the number of cells shedding and BVDV titer were in significant decrease at the same time interval. The gene SERPINF2 of the MDBK cells is successfully knocked out using CRISPR/Cas9 gene editing technology,and the SERPINF2 KO cells are established. Knockout of the gene SERPINF2 significantly inhibits BVDV replication.

Key words: SERPINF2, bovine viral diarrhea virus, CRISPR/Cas9

FU Qiang, CHEN Jun-zhen, GUO Yan-ting, YAO Gang, RAN Duo-liang, SHI Hui-jun. Effects of Knockout of Gene SERPINF2 via CRISPR/cas9 on the Replication of Bovine Viral Diarrhea Virus[J]. Biotechnology Bulletin, 2021, 37(5): 267-272.

Figures/Tables 8

Table 1 SgRNA sequences

向导RNA sgRNA	序列 Sequence （5'→3'）
SERPINF2	CACCGCATCCTGTCACCTCTGAGTG
SERPINF2	AAACCACTCAGAGGTGACAGGATGC
Scramble	CACCGCACTACCAGAGCTAACTCA
Scramble	aaacTGAGTTAGCTCTGGTAGTGC

Fig.1 Identification of lentiCRISPR V2 plasmid by enzyme digestion M: D2000 DNA marker, 1: enzyme cutting lentiCRISPR v2 vector

Fig.2 Efficiency Detection of SERPINF2 gene knockout in the MDBK cells WT：wild type, -：base deletion, +：base insertion, Number：base number, (Number)：Clone number of mutation such as deletion or insertion

Fig.3 Transcription levels of SERPINF2 mRNA in the MDBK cells infected by BVDV via qRT-PCR

Fig.4 qRT-PCR detection of BVDV RNA level in the SERPINF2 KO cells infected with BVDV

Fig.5 Detection of BVDV dsRNA accumulation in the SERPINF2 KO cells infected with BVDV by immunofluorescence staining

Fig.6 Detection results of virus titer in the SERPINF2 KO cells infected with BVDV

Fig.7 Detection results of cytopathic effect in the SERPINF2 KO cells infected with BVDV

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