Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells
Introduction
Baculovirus plays an important role as a natural agent controlling the size of insect populations (Jiang and Xia, 2014, Zhang et al., 2014a). However, it is a serious threat for beneficial insects such as silkworm, which is an important Lepidopteran model insect of economic value (Jiang and Xia, 2014). Since silkworm is the only host of Bombyx mori nucleopolyhedrovirus (BmNPV), viral resistance strains of this lepidopteran will benefit the silk industry. Thus far, a number of antiviral strategies have been used to create transgenic silkworms; these include RNA interference of the BmNPV core gene, overexpression of an endogenous or exogenous antiviral gene and regulation of host immunity. Current antiviral strategies only reduce viral replication and suppress the virus in infected cells and tissues but do not eliminate the viral genome from an infected silkworm.
Recent studies have described an efficient gene editing system targeting viral genomes with clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9) technology (Lijuan et al., 2015, Ma et al., 2014, Pellagatti et al., 2015). CRISPR/Cas9 is a component of the adaptive immune defense system in bacteria and archaea, and is used to defend against invading viruses and exogenous DNA (Ebina et al., 2013, Lijuan et al., 2015, Pellagatti et al., 2015). Recently, the CRISPR/Cas9 genome editing tool has been used widely for applied to the systematic investigate mammalian genomes in cell cultures (Ran et al., 2013, Shalem et al., 2014, Wang et al., 2014). This genome editing technology has great potential to edit animal genomes, and has been used for somatic genome editing (Chiou et al., 2015, Sanchez-Rivera et al., 2014, Wang et al., 2015), to correct genetic disorders (Meissner et al., 2014, Zou et al., 2015) and treat infectious diseases (Ebina et al., 2013, Ramanan et al., 2015, Seeger and Sohn, 2014, Tu et al., 2015, Yuan et al., 2015b). However, there are huge challenges in the large-scale application of the CRISPR/Cas9 genome editing system due to the high potential for off-target effects and homologous sequence repairs.
Recently, Ebina et al. reported that the CRISPR/Cas9 system can edit HIV-1 genome in human cells resulting in the removal of the viral DNA from host cells (Ebina et al., 2013, Sanyal et al., 2013). The technology has been widely applied in gene therapy. Several studies have successfully applied the CRISPR/Cas9 system to treat infectious diseases by disrupting viral genes, editing viral DNA and eliminating the virus genome (Ebina et al., 2013, Pellagatti et al., 2015). More importantly, the editing of human herpes virus, human papillomavirus and hepatitis B virus genomes with the CRISPR/Cas9 system has achieved remarkable success (Ebina et al., 2013, Liao et al., 2015, Seeger and Sohn, 2014, Yuan et al., 2015b). However, the CRISPR/Cas9 system has not been applied to counter viral infections in insects.
In the present study, we applied a highly efficient virus-inducible CRISPR/Cas9 technology to disrupt BmNPV in infected silkworm. First, we improved the CRISPR/Cas9 system gene editing efficiency by linking Cas9 and sgRNA expression cassettes in the same vector. Furthermore, to reduce the probability of off-target effects and the possible toxicity of CRISPR/Cas9 system induced by long-term expression of exogenous proteins in host cells, we optimized the gene editing system. Our results showed the successful construction of a CRISPR/Cas9 genome editing system with reduced potential off-target of effects and high editing efficiency that can be used for antiviral research in insects.
Section snippets
Cells and viruses
The B. mori cell line BmN-SWU1, was cultured at 27 °C in TC-100 medium (United States Biological, USA) supplemented with 10% (V/V) fetal bovine serum (FBS) (Gibco, USA) and 10% (V/V) penicillin/streptomycin (Pan et al., 2010). Recombinant BmNPV (vA4prm-EGFP) containing an EGFP marker gene driven by the B. mori actin A4 promoter was constructed previously (Dong et al., 2014, Zhang et al., 2014a). Viruses were cultured in BmN-SWU1 cells, and viral titers were determined by 50% tissue culture
Excision of the ie1 gene from BmNPV genome
Baculoviruses quickly establish a systemic infection in B. mori. Therefore, it is critical to rapidly disable viral replication in B. mori. For this purpose, we used the CRISPR/Cas9 system and the immediate early gene, ie-1, in BmNPV as the editing target. Besides, we linked all three expression cassettes (Cas9, U6-sgRNA and mCherry) into a single vector to improve the efficiency of the CRISPR/Cas9 system (Fig. 1A). After transfecting pIZ-Cas9-sgIE1-mCherry and pIZ-Cas9-sgMock-mCherry into
Discussion
Antiviral studies in silkworm are essential to improve not only sericulture production but also to control insect pests (Dong et al., 2014, Jiang and Xia, 2014, Zhang et al., 2014a). Thus far, disrupting the viral genome with the CRISPR/Cas9 gene editing system has not been previously reported in the baculovirus (Pellagatti et al., 2015, Wei et al., 2014). To apply the CRISPR/Cas9 system for insect pest control and to treat infectious diseases, we constructed a highly efficient gene editing
Acknowledgments
This work was supported by grants from the National High-tech R&D Program of China (No. 2013AA102507), the National Natural Science Foundation of China (Nos. 31272505, 31472152 and 31572466), China Agriculture Research System (CARS-22), China Postdoctoral Science Foundation (2015M582502) and Fundamental Research Funds for the Central Universities (XDJK2015D002).
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