hyperactive-piggybac-transposase-mediated-germline-transformation

Hyperactive <em>piggyBac</em> Transposase-mediated Germline Transformation in the Fall Armyworm, <em>Spodoptera frugiperda</em>

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic ...

University of Kentucky

RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects,  and , showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing , Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects.

Double-stranded RNA binding protein, Staufen, is required for the initiation of RNAi in coleopteran insects.

Doublesex (dsx), the downstream gene in the insect sex-determination pathway, is a key regulator of sexually dimorphic development and behavior across a variety of insects. Manipulating expression of dsx could be useful in the genetic control of insects. However, information on the sex-specific function of dsx in non-model insects is lacking.

Disruption of sex-specific doublesex exons results in male- and female-specific defects in the black cutworm, Agrotis ipsilon.

The functional information on heat-shock proteins (Hsp) and heat-shock promoters from an important agricultural insect pest, Spodoptera frugiperda, is still lacking. We conducted a genome-wide identification of Hsp genes and identified a total of 21 genes belonging to four major insect Hsp families (small heat-shock proteins, Hsp60, Hsp70, and Hsp90) in S. frugiperda. Expression of most of S. frugiperda (SfHsp) genes could be detected in Sf9 cells, embryos and larval tissues of S. frugiperda. The heat-inducible activity of heat-shock promoters from several SfHsp genes was tested in Sf9 cells and embryos. The promoter of SfHsp70D showed the high constitutive activity in cell line and embryos, while the activity of SfHsp20.15 and SfHsp20.71 promoters was most dramatically induced in Sf9 cells and embryos. In embryos, the heat-induced activity of SfHsp20.71 and SfHsp70D promoters outperformed commercially used ie1 and ie2 promoters. The heat-induced activity of SfHsp70D and SfHsp19.07 promoters were more robust than ie2 promoter in Sf9 cells. These SfHsp promoters with high basal activity or with heat-induced activity from low basal activity, could be used in S. frugiperda or other lepidopteran insects for many applications including transgenesis and genome editing.

Identification and functional analysis of promoters of heat-shock genes from the fall armyworm, Spodoptera frugiperda.

The cell lines derived from the fall armyworm (FAW), Spodoptera frugiperda, have been widely used for production of recombinant proteins for applications in both basic research and applications in medicine and agriculture. Promoters from the nucleopolyhedrovirus (NPV) are commonly used in these expression systems. These promoters have some limitations, which may be overcome by using promoters of genes from S. frugiperda. However, information on these promoters is not available. We identified several highly expressed genes from the transcriptomes of S. frugiperda midgut, fat body, epidermis, ovarian cell line (Sf9), and a midgut cell line (Sf17). The activity of potential promoters of 21 highly expressed genes was evaluated in Sf9 and Sf17 cells. Two of these promoters, SfHSC70-P1780 and SfPub-P2009, showed higher activity than commonly used hr5/ie1 (baculovirus enhancer element, hr5 and immediate early gene 1, ie1) promoter. Interestingly, the activity of these two promoters increased after adding hr5 enhancer element. The hr5/SfPub-P2009 promoter performance was evaluated by expressing an exogenous P450 protein in Sf9 cells using a plasmid-based expression system. The activity of this promoter was also evaluated in the FAW by expressing green fluorescence protein using the baculovirus expression system. In both cases, the hr5/SfPub-P2009 promoter performed better than the commonly used hr5/ie1 promoter. These strong endogenous promoters will be useful for studies in S. frugiperda and other lepidopteran insects for multiple applications, including protein expression, genome editing, and transgenic insects.

Identification and characterization of highly active promoters from the fall armyworm, Spodoptera frugiperda.

RNA interference (RNAi) is an important tool for gene function studies in insects, especially in non-model insects. This technology is also being developed for pest control. However, variable RNAi efficiency among insects is limiting its use in insects. Systemic RNAi in  requires systemic RNA interference defective protein 1 (. The expression of Ce in insect cell lines was shown to improve RNAi. However, the mechanisms through which this double-stranded RNA (dsRNA) transporter improves RNAi efficiency in insects is not known. We stably expressed  in two  cell lines, Sf9 and Sf17 cells derived from ovary and midgut, respectively. Expression of  enhanced RNAi efficiency in ovarian Sf9 cells, but not in midgut Sf17 cells. Reduced accumulation of dsRNA in late endosomes and successful processing dsRNA to siRNA contribute to enhanced RNAi efficiency in Sf9 cells. Transgenic  expressing  were produced and tested for RNAi efficiency. RNAi efficiency enhancement due to  expression showed tissue specificity. Compared to RNAi efficiency in wild-type  expressing transgenic  showed a significant improvement of RNAi in tissues such as Verson's glands. In contrast, no improvement in RNAi was observed in tissues such as midgut. The  cell-type specific and  tissue-specific enhancement of RNAi efficiency by  in  provides valuable information for improving RNAi in insects such as those belonging to order Lepidoptera where RNAi is variable and inefficient.

 systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner.

CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, , a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene,  (), and the juvenile hormone receptor,  (). PiggyBac transformation vectors were prepared to express sgRNAs targeting  and  under the control of the U6 promoter. Transgenic  expressing -sgRNA or -sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in -sgRNA and -sgRNA groups, respectively, showed mutations in their target genes.

Article	Year
Double-stranded RNA binding protein, Staufen, is required for the initiation of RNAi in coleopteran insects. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30061410	2018
Disruption of sex-specific doublesex exons results in male- and female-specific defects in the black cutworm, Agrotis ipsilon. Pest management science\| PubMed ID: 30520231	2019
Identification and functional analysis of promoters of heat-shock genes from the fall armyworm, Spodoptera frugiperda. Scientific reports\| PubMed ID: 32047182	2020
Identification and characterization of highly active promoters from the fall armyworm, Spodoptera frugiperda. Insect biochemistry and molecular biology\| PubMed ID: 32827641	2020
systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner. RNA biology\| PubMed ID: 33111632	2021
Expanding the Toolkit for Genome Editing in a Disease Vector, Transgenic Lines Expressing Cas9 and Single Guide RNA Induce Efficient Mutagenesis. The CRISPR journal\| PubMed ID: 33450159	2021

Xien Chen

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