key: cord-0723701-1wsmizn8
authors: Li, Guo; Wang, Xinjie; Liu, Yajing; Lv, Xinyuan; Li, Guanglei; Zhao, Chengcheng; Wang, Dandan; Huang, Xingxu; Hu, Xiaoxiang
title: Porcine reproductive and respiratory syndrome virus (PRRSV) inhibition with engineered Cas13d
date: 2020-03-17
journal: J Genet Genomics
DOI: 10.1016/j.jgg.2020.02.006
sha: 41a299cf667231ea83bcee42e0189433b632ad85
doc_id: 723701
cord_uid: 1wsmizn8

nan

and the CRISPR/Cas13b system has been reported to have the potent antiviral activity against 23 multiple single-strand RNA (ssRNA) viruses in the cell culture system (Freije et al., 2019) . A study 24 has shown that the efficiency of transcript knockdown mediated by Cas13d is significantly higher 25 than that of both Cas13a and Cas13b (Konermann et al., 2018) . 26

The CRISPR/Cas13 system could specifically bind and precisely cleave ssRNAs without 27 altering the host genome, providing a potential novel strategy against the foreign RNA viruses. In 28 this study, we aim to explore whether the CRISPR/Cas13d system can be used to inhibit the syndrome virus (PRRSV) as a model to test the hypothesis. Porcine reproductive and respiratory 31 syndrome (PRRS) caused by PRRSV is one of the most severe infectious diseases in the swine 32 industry, which leads to a huge economic loss worldwide (Chand et al., 2012; Zhou and Yang, 33 2010) . PPPSV is an enveloped, positive-strand RNA virus belonging to the Arteriviridae family in 34 the order of Nidovirales (Snijder et al., 2013) . 35 We constructed a reporter system to rapidly test the RNA cleavage efficiency of the 36 CRISPR/Cas13d system. The reporter vectors were composed of the mCherry fluorescence gene 37 fused with either PRRSV ORF4 or ORF5 under the control of a CMV promoter, with two nuclear 38 localization signals (NLSs) at the N-and C-termini of mCherry to localize the fusion proteins in the 39 nucleus ( Fig. S1A ). Seven 30-bp oligonucleotides, including one random sequence as the non-target 40 guide RNA (gRNA) control (NC-gRNA) and six gRNA sequences targeting ORF4 (ORF4-gRNAs) 41 or ORF5 (ORF5-gRNAs), were synthesized and cloned into gRNA-expression vectors, respectively 42 ( Fig. S1A ). The human codon-optimized Cas13d was synthesized and cloned into an expression 43 vector with two NLSs at its N-and C-termini and driven by the CMV promoter (named 44 NLS-Cas13d) (Fig. S1A) . A control vector containing a mutated Cas13d which lacks the 45 ribonuclease activity (named NLS-dCas13d) was also constructed ( NLS-Cas13d/ORF4-gRNAs was significantly decreased (Fig. 1A) . Further qRT-PCR analysis 52 revealed that the mRNA expression levels of mCherry-ORF4 were significantly decreased by 96%, 53 90%, and 95% by ORF4-gRNA1, ORF4-gRNA2, and ORF4-gRNA3, respectively (P < 0.0001; Fig.  54 efficiently knocked down the expression of mCherry-ORF4 or mCherry-ORF5 (90%) compared 60 with that combined with the NC-gRNA (P < 0.0001) (Fig. S1B) off-target events exist, we blasted ORF4-gRNA1 and ORF5-gRNA1 against the human reference 107 genome, and selected MAGI1 and NUP210 (having a high similarity with ORF4-gRNA1) and 108 5 dCas13d/gRNA-transfected cells (Fig. S3B) . Together, these results indicated the CRISPR/Cas13d 117 system used in this study has little or no significant off-target effects. 118 Viral infections underlie a variety of different diseases impacting both public health and 119 agricultural industries greatly. In this study, we focused on the antiviral capacity of the 120 CRISPR/Cas13d system and presented a new strategy for mammalian RNA virus interference. 121

Interference efficiency, specificity, and applicability are the main concerns of RNA virus editing 122 tools. Our results showed that NLS-Cas13d combined ORF4-gRNA1 or ORF5-gRNA1 cleaved 96% 123 of mCherry-ORF4 mRNA or 84% of mCherry-ORF5 mRNA (Fig. 1B) . The efficiency is equal to a 124 previous report that Cas13d mediated a 97% knockdown of RNA viruses in Arabidopsis 125 

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