Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells

Dong, Zhanqi; Chen, Tingting; Zhang, Jun; Hu, Nan; Cao, Mengji; Dong, Fangyan; Jiang, Yaming; Chen, Peng; Lü, Cheng; Pan, Min‐Hui

doi:10.1016/j.antiviral.2016.03.009

Cited by 51 publications

(40 citation statements)

References 35 publications

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“…However, silkworm disease caused annually by BmNPV results in huge economic losses in the sericulture industry (Jiang and Xia, ). RNAi and overexpression of extraneous proteins have been used to generate transgenic antiviral silkworm lines as a potential therapeutic approach (Zhang, He et al , ; Dong et al , ). However, it is difficult to significantly increase the antiviral effect, which hinders its application in sericulture and in conducting further in‐depth studies.…”

Section: Discussionmentioning

confidence: 99%

“…We selected the two target sites of the BmNPV ie‐0 and ie‐2 genes as gene editing sites, which were located at ie‐0 and ie‐2 gene transcription start sites 304 and 426 respectively in the BmNPV genome. The two target sgRNA sites were ligated in to the pSL1180‐U6‐sgRNA vector at the Bbs I restriction enzyme site (Dong et al , ). The guide RNA target sequence was controlled by the U6 promoter, and the TTTTTT sequence acts as a guide RNA termination signal.…”

Section: Experimetal Proceduresmentioning

confidence: 99%

“…In 2016, we first successfully edited the BmNPV immediate early‐1 (IE‐1) gene in silkworm cells and proved that the system could effectively suppress virus replication by establishing a Cas9 stable cell line. Establishing a virus‐induced CRISPR/Cas9 system could further reduce host toxicity and off‐target efficiency (Dong et al , ). In silkworm, an antiviral effect has also been achieved by editing the ie‐1 and me‐53 genes of the BmNPV in a transgenic CRISPR system (Chen et al , ).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Dong

et al. 2018

Insect Molecular Biology

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The CRISPR/Cas9 system is a powerful tool for the treatment of infectious diseases. In our previous study, we knocked out the Bombyx mori nucleopolyhedrovirus (BmNPV) key genes and BmNPV-dependent host factor to generate transgenic antiviral strains. To further expand the range of target genes for BmNPV and more effectively prevent and control pathogenic infections, we performed gene editing and antiviral analysis by constructing a target-directed baculovirus early transcriptional activator immediate early-0 (ie-0) and 2 (ie-2) transgenic silkworm line. We hybridized it with Cas9 transgenic line to produce a double-positive transgenic Cas9(+)/sgIE0-sgIE2(+) line that could activate the CRISPR gene editing system. We first demonstrated that the system is capable of efficiently editing target genes and resulting in fragment deletions in the BmNPV genome. Survival rate of the transgenic Cas9(+)/sgIE0-sgIE2(+) line reached 65% after inoculation with 1 × 10 occlusion bodies/larva. Molecular analysis showed that BmNPV DNA replication and viral gene expression level in the transgenic Cas9(+)/sgIE0-sgIE2(+) line were significantly inhibited compared with the control Cas9(-)/sgIE0-sgIE2(-) line. These results indicated that IE-0 and IE-2, as baculovirus early transcriptional activators, can be used as target sites for gene therapy and that multigene editing could expand the range of target sites for research to create silkworm resistance breeds.

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Section: Discussionmentioning

confidence: 99%

Section: Experimetal Proceduresmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Dong

et al. 2018

Insect Molecular Biology

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“…After 24 hours, cells were infected with BmNPV at an MOI of 1. At the indicated time points, cells were harvested and total RNA was prepared using TRIzol reagent (Invitrogen, USA) as described previously43. Samples were normalized to total RNA and reverse transcription reactions were performed with SYBR Select Master Mix Reagent (Bio-Rad, USA) using specific primers (S1 Table).…”

Section: Methodsmentioning

confidence: 99%

“…The cells were stained with a monoclonal α-ATAD3A or α-HSPD1 antibody and stained with Alexa 555-conjugated goat anti-mouse IgG, FITC-conjugated goat anti-mouse IgG, Hoechst 33258 and Mito-Tracker Green (Beyotime, China) as described above43.…”

Section: Methodsmentioning

confidence: 99%

Baculovirus LEF-11 Hijack Host ATPase ATAD3A to Promote Virus Multiplication in Bombyx mori cells

Dong

et al. 2017

Sci Rep

Self Cite

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Research on molecular mechanisms that viruses use to regulate the host apparatus is important in virus infection control and antiviral therapy exploration. Our previous research showed that the Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 localized to dense regions of the cell nucleus and is required for viral DNA replication. Herein, we examined the mechanism of LEF-11 on BmNPV multiplication and demonstrated that baculovirus LEF-11 interacts with Bombyx mori ATAD3A and HSPD1 (HSP60) protein. Furthermore, we showed that LEF-11 has the ability to induce and up-regulate the expression of ATAD3A and HSPD1, phenomena that were both reversed upon knockdown of lef-11. Our findings showed that ATAD3A and HSPD1 were necessary and contributed to BmNPV multiplication in Bombyx mori cells. Moreover, ATAD3A was found to directly interact with HSPD1. Interestingly, ATAD3A was required for the expression of HSPD1, while the knockdown of HSPD1 had no obvious effect on the expression level of ATAD3A. Taken together, the data presented in the current study demonstrated that baculovirus LEF-11 hijacks the host ATPase family members, ATAD3A and HSPD1, efficiently promote the multiplication of the virus. This study furthers our understanding of how baculovirus modulates energy metabolism of the host and provides a new insight into the molecular mechanisms of antiviral research.

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A comprehensive overview of CRISPR/Cas 9 technology and application thereof in drug discovery

Khurana

Sayed

Singh

et al. 2022

J of Cellular Biochemistry

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Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)‐Cas technology possesses revolutionary potential to positively affect various domains of drug discovery. It has initiated a rise in the area of genetic engineering and its advantages range from classical science to translational medicine. These genome editing systems have given a new dimension to our capabilities to alter, detect and annotate specified gene sequences. Moreover, the ease, robustness and adaptability of the CRISPR/Cas9 technology have led to its extensive utilization in research areas in such a short period of time. The applications include the development of model cell lines, understanding disease mechanisms, discovering disease targets, developing transgenic animals and plants, and transcriptional modulation. Further, the technology is rapidly growing; hence, an overlook of progressive success is crucial. This review presents the current status of the CRISPR–Cas technology in a tailor‐made format from its discovery to several advancements for drug discovery alongwith future trends associated with possibilities and hurdles including ethical concerns.

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Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells

Cited by 51 publications

References 35 publications

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

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

Baculovirus LEF-11 Hijack Host ATPase ATAD3A to Promote Virus Multiplication in Bombyx mori cells

A comprehensive overview of CRISPR/Cas 9 technology and application thereof in drug discovery

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