Transgenic CRISPR/Cas9-mediated viral gene targeting for antiviral therapy of Bombyx mori

43 Pages·2017·3.82 MB·English

JVI Accepted Manuscript Posted Online 25 January 2017
J. Virol. doi:10.1128/JVI.02465-16
Copyright © 2017 Chen et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
1 Transgenic CRISPR/Cas9-mediated viral gene targeting for antiviral therapy of
2 Bombyx mori nucleopolyhedrovirus (BmNPV)
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4 Shuqing Chena,b,1, Chengxiang Houc,1, Honglun Bia, Yueqiang Wanga, Jun Xua,b,
5 Muwang Lic, Anthony A. Jamesd, Yongping Huanga,2, Anjiang Tana,2 D
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6 aKey Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant loa
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7 Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese d

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8 Academy of Sciences, Shanghai 200032, China. m

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9 bUniversity of Chinese Academy of Sciences, Beijing 100049, China :/
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10 cSericultural Research Institute, Jiangsu University of Science and Technology, sm
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11 Zhenjiang 212018, Jiangsu, China g
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12 dDepartments of Microbiology & Molecular Genetics and Molecular Biology & M
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13 Biochemistry, University of California, Irvine, CA 92697-3900， USA rc
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15 1Shuqing Chen and Chengxiang Hou contributed equally to this work. 1
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16 2Corresponding author: Yongping Huang and Anjiang Tan g
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17 Key Laboratory of Insect Developmental and Evolutionary Biology,
18 Institute of Plant Physiology and Ecology,
19 Shanghai Institutes for Biological Sciences,
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20 Chinese Academy of Sciences,
21 300 Feng Lin Road, Shanghai 200032, China
22 Phone: [phone]
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23 E-mail: [email protected] or [email protected] o
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24 a
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25 Running title: Transgenic CRISPR/Cas9 for antiviral therapy of BmNPV ro
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26 tp
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27 Word counts: a
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28 Abstract: 119 rg
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29 Significance: 72 M
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30 Text: 4165 3
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32 Abstract
33 We developed a novel anti-viral strategy by combining transposon-based transgenesis
34 and the clustered regularly interspaced short palindromic repeats
35 (CRISPR)/CRISPR-associated 9 (Cas9) system for direct cleavage of the Bombyx
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36 mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in w
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37 silkworms. We demonstrate that transgenic silkworms constitutively-expressing Cas9 loa
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38 and guide RNAs (gRNAs) targeting the BmNPV immediate early-1 (ie-1) gene and d

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39 me53 gene effectively induce target-specific cleavage and subsequent mutagenesis, m

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40 especially large segment deletions (~ 7 kilobase-pairs [kb]) in BmNPV genomes and tp
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41 thus exhibit a robust suppression of BmNPV proliferation. Transgenic animals i.
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42 exhibited higher and inheritable resistance to BmNPV infection when compared to
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43 wild-type animals. Our approach will not only contribute to modern sericulture but /

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44 also shed light on future anti-viral therapy. M
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45 Importance 3
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46 Pathogen genome targeting has shown its potential in anti-viral research. However, 1
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47 transgenic clustered regularly interspaced short palindromic repeats y

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48 (CRISPR)/CRISPR-associated 9 (Cas9) system-mediated viral genome targeting has e
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49 not been reported as an antiviral strategy in a natural animal host of a virus. Our data
50 provide an effective approach against Bombyx mori nucleopolyhedrovirus (BmNPV)
51 infection in a real-world biological system and demonstrate the potential of transgenic
52 CRISPR/Cas9 systems in anti-viral research in other species.
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54 1. Introduction
55 The silkworm, Bombyx mori, a completely domesticated and economically important
56 insect, is susceptible to a variety of diseases. In China, almost 80% of silk cocoon
57 crop losses are attributed to viral diseases (1). Bombyx mori nucleopolyhedrovirus
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58 (BmNPV) is a major viral pathogen that causes substantial economic losses to mass w
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59 silkworm rearing in silkworm-raising countries (1, 2), and it still remains a big loa
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60 challenge to the sericulture industry without effective prevention methods. BmNPV is d

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61 a member of the Baculoviridae family, which is characterized by a rod-shaped, m

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62 enveloped virion containing a closed, circular, double-stranded DNA (dsDNA) tp
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63 genome of ~130 kilobase-pairs (kb) in length. Moving from a time- and i.
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64 labor-consuming traditional breeding approach, numerous transgenic strategies have
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65 been developed in modern sericulture to improve BmNPV resistance, although their /

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66 practical use remains to be established. These transgenic methods are divided into two M
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67 main types: overexpression of endogenous or exogenous anti-viral genes (3, 4), or h

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68 inhibition of viral genes through RNA interference (RNAi) (5, 6). Limited success has ,

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69 been achieved with the overexpression strategy. Only two antiviral genes, the 9

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70 endogenous antiviral genes Bmlipase-1 and the exogenous antiviral genes hycu-ep32,
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71 have been reported to enhance the resistance of silkworms to BmNPV infection (3, 4). s
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72 In contrast, transgenic RNAi has been applied extensively by silencing one or
73 multiple BmNPV essential genes to destroy specific viral mRNAs (7, 8). It is
74 important to select appropriate genes as targets in RNAi-based anti-BmNPV
75 experiments as silencing different BmNPV genes results in variable anti-viral
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76 resistance efficiency (9). Overall, moderate virus resistance has been reported for
77 transgenic RNAi-based anti-BmNPV strategies, thus leaving the major challenge of
78 how to inhibit viral DNA replication due to persistence of BmNPV genomic DNA.
79 Recent advances in gene therapy strategies support pathogen genome targeting as a
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80 promising strategy in anti-viral research. In contrast to the RNAi strategy that works w
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81 at a post-transcriptional level, pathogen genome targeting aims at directly disrupting loa
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82 pathology-causing viral DNA or RNA that persists or accumulates in host cells, thus d

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83 eliminating viruses in the host. The clustered regularly interspaced short palindromic m

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84 repeats (CRISPR)/CRISPR-associated (Cas) system, which has several advantages tp
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85 over homing endonucleases (HEs), zinc finger nucleases (ZFNs) and transcription i.
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86 activator-like effector nucleases (TALENs), has greatly facilitated the pathogen
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87 genome targeting strategy (10). The CRISPR/Cas system has been utilized for /

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88 efficiently eradicating viruses in cell culture and mouse model of human disease, M
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89 including the hepatitis B virus (HBV) (11, 12), Epstein-Barr virus (EBV) (13, 14) and h

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90 human immunodeficiency virus (HIV)-1 in both pre-integration and provirus stages ,

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91 (15, 16), providing initial evidence for efficacy of CRISPR/Cas9 system-mediated 9

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92 antiviral therapy. The development of the RNA-targeting CRISPR/Cas system has
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93 expanded the scope of genetic engineering of pathogens to target RNA viruses, which s
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94 have no DNA stages in their life cycles (17). Although a facile and efficient
95 alternative to ZFNs and TALENs, CRISPR/Cas system was reported to induce a
96 high-frequency off-target mutagenesis (18). Several approaches have been developed
97 to improve the specificity of the gRNA:Cas9 tool and accelerate its use for therapeutic
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98 applications (19-22), but more tests in vivo need to be conducted for a better
99 understanding and non-biased assessment of applicability. Moreover, it is important
100 and of general interest to demonstrate the efficacy of CRISPR/Cas9 system-mediated
101 antiviral therapy in a legitimate, natural host-virus interaction.
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102 We show here that a transgenic CRISPR/Cas9 system can be used as an w
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103 anti-BmNPV strategy in a real-world biological system. We constructed a piggyBac loa
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104 plasmid that encodes a Cas9 protein under the control of a constitutive baculovirus d

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105 IE1 promoter and guide RNA (gRNA) expression cassettes under the control of U6 m

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106 promoters. The immediate early-1 (ie-1) gene and the me53 gene of BmNPV were tp
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107 selected as the target genes, and two gRNAs were designed to target each of them i.
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108 separately. Transgenic silkworms carrying such a construct showed significant
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109 improvement in viral resistance with a significant reduction in both viral DNA copy /

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110 numbers and gene expression after inoculation with BmNPV occlusion bodies (OB). M
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111 This report of successful in vivo anti-viral research using the transgenic CRISPR/Cas9 h

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112 system in the context of natural viral infection in animals contributes to modern ,

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113 sericulture and provides the basis for future anti-viral therapy in other species. 9

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114 2. Materials and Methods u
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115 Silkworm strain and virus stock
116 The multivoltine, non-diapausing Nistari strain was used in all experiments. Larvae
117 were reared on fresh mulberry leaves under standard conditions. The wild-type
118 Zhejiang strain of BmNPV (GenBank accession JQ991008) was propagated in fifth
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119 instar larvae and used for infection. The OBs were harvested from the infected
120 hemolymph before the larvae died. The virus stock was prepared as described
121 previously (23). The number of OBs were counted using Neubauer hemocytometer.
122 The virus stock, at a concentration of 109 OBs/ml, was diluted serially to prepare
123 inocula with concentrations of 108 OBs/ml, 107 OBs/ml and 106 OBs/ml. D
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124 Target gene selection and vector construction a
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125 The ie-1 gene is essential for Autographa californica multiple nucleopolyhedrovirus fr
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126 (AcMNPV) DNA replication (24), and the me53 gene is required for efficient budded h
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127 viruses (BV) production (25, 26). Both genes were reported to be transcriptionally :/
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128 active as early as 1 hour post-infection (hpi) in AcMNPV (27).. We selected these two a
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129 genes in BmNPV as the target genes since they are major early-transcribed genes that o
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130 are involved with baculovirus propagation, and they are separated by a distance of ~5 o
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131 kb in the BmNPV genome which intended to produce large fragment deletions by the a
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132 CRISPR/Cas9 machinery (28). We searched ie-1 and me53 gene sequences in 3
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133 BmNPV genome for potential 20-base targeting sequences and chose four candidates 2
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134 that are compatible with consensus gRNA target sites (GN19NGG): ie1-s1:
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135 5’-GTGAACCAACCATCGGCAACTGG-3’ and ie1-s2: u
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136 5’-GAGTCAAATATTAAAGTATTCGG-3’ for ie-1 gene; me53-s1: t
137 5’-CCGCCGCGTCGGGTCTGGTGCAC-3’ and me53-s2:
138 5’-CCTAATTATTTCAAACACTTTGC-3’ for me53 gene. To avoid unintended gene
139 disruptions in silkworms, we blasted the four targeting sequences with the silkworm
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140 genome in KAIKObase (http://sgp.dna.affrc.go.jp/KAIKObase/). The potential
141 genomic off-target loci that were most similar to the gRNA targeting sequences had
142 more than three mismatches, indicating these targeting sequences can be used for
143 BmNPV targeting in silkworms (29). A constitutive Cas9 expression cassette under
144 the control of a baculovirus immediate-early gene promoter IE1 and four targeting D
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145 cassettes expressing the gRNAs targeting ie-1 and me53 under the separate control of nlo
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146 the silkworm small nuclear RNA promoter (U6 promoter, a ubiquitous polymerase III e
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147 promoter derived from the upstream sequence of the B. mori U6-2 small nuclear RNA ro
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148 gene) were constructed through a series of cloning steps to generate t
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149 pBac-EGFP-Cas9/4×gRNAs. The cloning of IE1-Cas9 and U6 promoter was carried /jv
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150 out as described previously (30-32). All primers used for the vector construction are s
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151 listed in Table 1. r
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152 Silkworm genetic transformation M
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153 DNA solutions containing pBac-EGFP-Cas9/4×gRNAs and the helper plasmids (33) 3
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154 were microinjected into preblastoderm G0 embryos that were then incubated at 25 °C 20
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155 in a humidified chamber for 10-12 d until larval hatching. Larvae were reared on fresh
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156 mulberry leaves under standard conditions. Putative transgenic (TG) G0 adults were u
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157 mated to wild-type (WT) moths to produce G1 progenies, and G1 progenies were t
158 scored for the presence of the marker gene EGFP using fluorescence microscopy
159 (Nikon AZ100). Inverse PCR was carried out as described previously to investigate
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160 genomic insertion loci of the transgene (34), and the primers used for detection are
161 listed in Table 1.
162 Off-target assays
163 To evaluate the off-target effects in TG silkworms, we selected three exonic off-target D
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164 sequences with perfect matches for the seed sequence (8-13 nt) adjacent to the PAM n
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165 using CRSPRdirect (http://crispr.dbcls.jp/) (35) and KAIKObase for each of the four d
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166 target sites. TG silkworm genomic DNA was used as a substrate to amplify putative fr
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167 off-target regions, and amplicons were cloned, sequenced and compared to the WT h
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168 silkworm genome. The off-target candidates are listed in Table 2 and primer :/
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169 sequences used for detection are listed in Table 1. a
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170 Viral inoculation and mortality analyses g
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171 Newly exuviated third instar larvae were starved for 12 hrs before viral inoculation.
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172 Each larva was fed a 1 cm2 piece of mulberry leaf that had been smeared with 10 µl of h

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173 an OB suspension. Only those larvae that consumed the entire leaf piece after 24 hrs ,
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174 of feeding were maintained. Virus-fed larvae were reared subsequently on fresh 9

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175 mulberry leaves and observed for signs of infection. Dead larvae were removed g
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176 immediately to prevent horizontal contamination. Mortality was recorded daily until t
177 the 10 days post-infection (dpi). The TG lines were mated with WT to generate
178 heterozygous offspring for viral inoculation. The resistance of each TG line was
179 investigated via oral infection with wild BmNPV using four different polyhedra doses,
180 107, 106, 105 and 104 OBs/larva, achieved by applying 10 µl of OB suspensions with
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181 concentrations of 109 OBs/ml, 108 OBs/ml, 107 OBs/ml and 106 OBs/ml on a fresh
182 mulberry leaf. In each infected group of a TG line, 60 newly exuviated third instar TG
183 larvae were each provided a leaf and monitored to ensure they consumed it
184 completely. The non-infected group (0 OBs/larva), also including 60 larvae, was fed
185 on leaves applied with an equal volume of sterile water. WT larvae were treated in D
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186 parallel as the control. nlo
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187 Transmission electron microscopy d

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188 Midguts dissected from 106 OBs/larva treated TG-A, TG-B, TG-C and WT silkworms h
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189 at 60 hpi were cut into 1-2 mm sized midgut fragments which were then fixed with :/
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190 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) overnight at 4°C; washed a
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191 with phosphate buffer and post-fixed with 1% osmium tetroxide in phosphate buffer o
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192 for 1-2 h; washed again, dehydrated by a graded series of ethanol and then embedded o
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193 in Embed812 resin. Ultrathin sections (60-90nm) were stained in 2% uranyl acetate a
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194 (pH 5.0), followed by 10 mM lead citrate (pH 12) and viewed with a transmission 3
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195 electron microscope, Hitachi H-7650. The midguts from more than six larvae were cut 2
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196 for each group.
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197 qPCR analysis of virus real-time accumulation levels after BmNPV oral infection e
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198 The virus real-time accumulation level was measured by viral DNA quantification.
199 The whole larvae samples were separately collected from 106 OBs/larva-treated
200 silkworms in the TG-A, TG-B, TG-C and WT group (six larvae as a sample in each
201 group) once every 12 hrs (from 0 to 72 hpi) and ground with tissue lyser before DNA
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