CRISPR/Cas9‐mediated disruption of the immediate early‐0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm

Dong, Zhanqi; Hu, Zhigang; Qi, Qin; Dong, Fangyan; Huang, Liang; Long, Jiangqiong; Chen, P.; Lü, Cheng; Pan, Min‐Hui

doi:10.1111/imb.12529

Cited by 23 publications

(20 citation statements)

References 38 publications

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“…The CRISPR/Cas9 gene editing technology has also been used in antiviral resistance breeding by editing host factor and viral key genes in BmNPV infection. However, the antiviral resistance level using this system has now reached a plateau [13, 16, 17].…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cpf1 mediated genome editing enhancesBombyx moriresistance to BmNPV

Dong

et al. 2020

Preprint

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18CRISPR/Cas12a (Cpf1) is a single RNA-guided endonuclease that provides new 19 opportunities for targeted genome engineering through the CRISPR/Cas9 system. Only 20 AsCpf1 have been developed for insect genome editing, and the novel Cas12a orthologs 21 nucleases and editing efficiency require more study in insect. We compared three 22 Cas12a orthologs nucleases, AsCpf1, FnCpf1, and LbCpf1, for their editing efficiencies 23 and antiviral abilities in vitro. The three Cpf1 efficiently edited the BmNPV genome 24 and inhibited BmNPV replication in BmN-SWU1 cells. The antiviral ability of the 25 FnCpf1 system was more efficient than the SpCas9 system after infection by BmNPV. 26 We created FnCpf1×gIE1 and SpCas9×sgIE1 transgenic hybrid lines and evaluated the 27 gene editing efficiency of different systems at the same target site. We improved the 28 antiviral ability using the FnCpf1 system in transgenic silkworm. This study 29 demonstrated use of the CRISPR/Cpf1 system to achieve high editing efficiencies in 30 the silkworm, and illustrates the use of this technology for increasing disease resistance. 31Genome editing is a powerful tool that has been widely used in gene function, gene 36 therapy, pest control, and disease-resistant engineering in most parts of pathogens 37 research. Since the establishment of CRISPR/Cas9, powerful strategies for antiviral 38 therapy of transgenic silkworm have emerged. Nevertheless, there is still room to 39 expand the scope of genome editing tool for further application to improve antiviral 40 research. Here, we demonstrate that three Cpf1 endonuclease can be used efficiency 41 editing BmNPV genome in vitro and in vivo for the first time. More importantly, this 42 Cpf1 system could improve the resistance of transgenic silkworms to BmNPV compare 43 with Cas9 system, and no significant cocoons difference was observed between 44 transgenic lines infected with BmNPV and control. These broaden the range of 45 application of CRISPR for novel genome editing methods in silkworm and also enable 46 sheds light on antiviral therapy.47 49 Genome editing introduces DNA mutations in the form of insertions, deletions or 50 base substitutions within selected DNA sequences [1]. Clustered regularly interspaced 51 short palindromic repeats (CRISPR) gene editing technology has been used in gene 52 function research, genetic improvement, modelling biology and gene therapy [2-5]. 53 Three effector proteins of class 2 type V CRISPR systems, the CRISPR/CRISPR-54 associated 12a (Cas12a, known as Cpf1) proteins of Lachnosperaceae bacterium 55 (LbCpf1), Francisella novicida (FnCpf1) and Acidaminoccocus sp. (AsCpf1), have 56 been shown to efficiently edit mammalian cell genomes with more efficient genome 57 editing than the widely used Streptococcus pyogenes Cas9 (SpCas9) [6-8]. However, 58 the CRISPR/Cpf1 system was rarely used for insect genome editing research and 59 antiviral therapy [9].60 The silk industry (Bombyx mori, B. mori), suffers great economic losses due to B. 61 mori nucleopolyhedr...

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Section: Introductionmentioning

confidence: 99%

CRISPR/Cpf1 mediated genome editing enhancesBombyx moriresistance to BmNPV

Dong

et al. 2020

Preprint

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“…Bombyx mori , which is not only an important economic insect (Dong et al ., ) but also a significant model insect, plays essential roles in the scientific research of insect physiology, development, genetics and immune regulation (Sun et al ., ; Xu et al ., ; Yu et al ., ; Zhang et al ., ; Zhou et al ., ). Insect storage proteins are haemolymph‐specific proteins that accumulate to their highest concentrations in the final instar.…”

Section: Introductionmentioning

confidence: 99%

Expressional analysis of the silkworm storage protein 1 and identification of its interacting proteins

Geng

et al. 2019

Insect Molecular Biology

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Storage proteins are haemolymph‐specific proteins in insects, mainly synthesized in the fat body, released into the haemolymph, and then selectively reabsorbed by the fat body before pupation. These storage proteins play an important role in insect metamorphosis and egg development. Some of these storage proteins are responsive to pathogen infection and can even suppress pathogen multiplication. However, the mechanisms of the physiological, biochemical and immune‐responsive functions of storage proteins remain unclear. In this study, the expression patterns of Bombyx mori storage protein 1 (BmSP1) during the larval stage were analysed. Then, BmSP1 protein fused with enhanced green fluorescent protein (EGFP) was successfully expressed in a B. mori baculovirus vector expression system. Quantitative real‐time PCR showed that the expression level of BmSP1 increased with the advance of instars and reached the highest level in the fifth instar, especially in the fat body. Recombinant BmSP1 expressed in silkworm larvae inhibited haemolymph melanization. Then, proteins that interact with BmSP1 were identified with EGFP used as an antigenic determinant by co‐immunoprecipitation. A 30 kDa low molecular weight lipoprotein PBMHP‐6 precursor (BmLP6) was shown to interact with BmSP1. Yeast two‐hybrid experiments confirmed the interaction between BmSP1 and BmLP6. The results obtained in this study will be helpful for further study of the functions of BmSP1 and BmLP6 in the regulatory network of silkworm development and innate immunity.

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“…An intriguing new strategy that can eradicate virus replication using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9) genome editing technology has been widely used in pathogenic viruses including human immunodeficiency virus 1 (HIV-1), human papillomavirus, hepatitis B virus (HBV), Epstein-Barr virus, plant virus, or flaviviruses in infected cells or animal models [10,11]. In the past 4 years, Cas9 technology has been extensively utilized in basic science and transgenic breeding settings to treat genetic diseases and infectious diseases in silkworm [12][13][14]. Our laboratory has employed CRISPR/Cas9 technology and developed a BmNPV specific gene editing system to excise the BmNPV genome ie-1 gene from infected cells of Bombyx mori for the first time [15].…”

Section: Introductionmentioning

confidence: 99%

“…Since 2017, many of antiviral strains, including gene editing of ie-0, ie-1, ie-2, or me53 genes, have been cultivated through transgenic breeding mainly through strategies of signal gene editing, double gene editing, and large fragment deletion [12,13]. At the same time, we have established a virus-inducible CRISPR/Cas9 system that can reduce the off-target efficiency and host toxicity of gene editing systems.…”

Section: Introductionmentioning

confidence: 99%

“…All of these antiviral strains have achieved remarkable antiviral effects through transgenic genome editing. However, the introduction of nucleases into cells by these nuclease-based genome editing methods is still inefficient and has partial selectivity of viral gene editing for removing the entire BmNPV genome [13,14]. Therefore, there is an urgent need to screen and identify BmNPV genes and used them to improve the antiviral viability by multi-gene editing in the BmNPV genome.…”

Section: Introductionmentioning

confidence: 99%

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A Multiplex CRISPR/Cas9 System for Use as an Anti-BmNPV Therapeutic

Dong¹,

Q²,

Hu³

et al. 2018

Preprint

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Clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology guided by a single-guide RNA (sgRNA) has recently opened a new avenue for antiviral therapy. A unique capability of the CRISPR/Cas9 system is multiple genome engineering. However, there are few applications in insect viruses by a single Cas9 enzyme targeting two or more sgRNA at different genomic sites for simultaneous production of multiple DNA breaks. To address the need for multi-gene editing and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system to express multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells. Here, ie-1, gp64, lef-11, and dnapol genes were screened and identified as multiple sgRNA editing sites according to the BmNPV system infection and DNA replication mechanism. Furthermore, we constructed a multiplex editing vector sgMultiple to efficiently regulate multiplex gene editing steps and inhibit BmNPV replication after viral infection. This is the first report that describes the application of multiplex CRISPR/Cas9 system inhibiting insect virus replication. This multiplex system can significant enable the potential of CRISPR/Cas9-based multiplex genome engineering in transgenic silkworms.

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CRISPR/Cas9‐mediated disruption of the immediate early‐0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm

Cited by 23 publications

References 38 publications

CRISPR/Cpf1 mediated genome editing enhancesBombyx moriresistance to BmNPV

CRISPR/Cpf1 mediated genome editing enhancesBombyx moriresistance to BmNPV

Expressional analysis of the silkworm storage protein 1 and identification of its interacting proteins

A Multiplex CRISPR/Cas9 System for Use as an Anti-BmNPV Therapeutic

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