THE FALL ARMYWORM FUNCTIONAL GENOMICS:GENOME EDITING AND RNAI

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1018897
• Grant No.: 2019-67013-29351
• Cumulative Award Amt.: $453,997.00
• Proposal No.: 2018-07651
• Project Start Date: Mar 15, 2019
• Project End Date: Mar 14, 2023
• Grant Year: 2019
• Program Code: [A1112]- Pests and Beneficial Species in Agricultural Production Systems

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Entomology

Non Technical Summary
The fall armyworm (FAW) is a polyphagous pest that causes economic losses to many crops.Voracious feeding, year-round breeding and highly efficient detoxification system to cope withplant toxins and insecticides make this a major international pest. Therefore, there is a need to studythe molecular basis of biological processes that make this insect a successful invader and pest. Oneof the reasons for the lack of functional genomics information on this insect is its poor response toRNA interference (RNAi). The CRISPR/Cas9 genome editing method could help to overcomethese limitations. Preliminary experiments showed that CRISPR/Cas9 system works in FAW. Themajor goal of this proposal is to develop the CRISPR/Cas9 system for efficient knockout of genesin FAW. To be able to identify mutants in G0, methods based on multiple sgRNAs in combinationwith marker sgRNA will be developed. The CRISPR/Cas9 system will be employed to improveRNAi efficiency in FAW by mutating dsRNase genes and by expressing SID-1 dsRNA transportergene from Caenorhabditis elegans and StaufenC gene from Leptinotarsa decelimineata, the twogenes shown to be required for efficient RNAi in nematodes and beetles respectively. The proposedresearch will develop CRISPR/Cas9 and RNAi methods that could help in functional genomicsresearch leading to the development of novel and efficient methods to control FAW.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
216	3110	1040	100%

Knowledge Area
216 - Integrated Pest Management Systems;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology;

Keywords

editing

fall armyworm

gene

rna interference

Goals / Objectives
The major goal of this proposal is to develop the CRISPR/Cas9 system for efficient knockout of genesin FAW. To be able to identify mutants in G0, methods based on multiple sgRNAs in combinationwith marker sgRNA will be developed. The CRISPR/Cas9 system will be employed to improveRNAi efficiency in FAW by mutating dsRNase genes and by expressing SID-1 dsRNA transportergene from Caenorhabditis elegans and StaufenC gene from Leptinotarsa decelimineata, the twogenes shown to be required for efficient RNAi in nematodes and beetles respectively.

Project Methods
The main objectives of this proposal are to optimize various parameters to improve this system sothat it could be used as a routine functional genomics method in FAW and other lepidopteranpests. To increase the frequency of gene knockout individuals in G0, we will optimizeparameters including treatment of eggs, the timing of injection, injection of eggs, ratio of guideRNA and Cas9, rearing, and screening of offspring developed from injected eggs, evaluation ofsomatic mosaics and genotyping of offspring. We will also try multiple guide RNA-basedmethods to increase the editing frequency to be able to generate enough knockouts to evaluategene function in G0. Research in mammals showed that multiple guide RNAs enhanceCRISPR/Cas9 mediated knockout efficiency [123]. We will also develop a method tosimultaneously edit two genes, a marker gene and a target gene to facilitate identificationmutants of genes (e.g. dsRNase) with no visible phenotypes. We will investigate the potentialoff-target effects caused by single and multiple guide RNAs in FAW. The potential for off-targeteffects by sgRNAs that show 1, 2, and 3 mismatches in the seed region with the non-target genesin the genome will be evaluated by T7E1 assay and sequencing of PCR products. We will use theCRISPR/Cas9 system developed to produce FAW transgenic lines with improved RNAi that canbe used in functional genomics studies. The genes coding for dsRNases and REase will beknocked out and their contribution to RNase activity and RNAi inefficiency will be evaluated.We will also produce transgenic FAW lines expressing transporter gene from C. elegansandStaufenC gene from Leptinotarsa decelimineata to test if these genes improve RNAi.

Progress 03/15/19 to 03/14/23

Outputs
Target Audience:Researchers working on Fall armyworm Control. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Post-doctoral fellows were trained. How have the results been disseminated to communities of interest?Through publishing in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Developing safe and effective double-stranded RNA (dsRNA) delivery systems remains a major challenge for gene silencing, especially in lepidopteran insects. This study evaluated the protamine sulfate (PS)/lipid/dsRNA nanoparticle (NP) delivery system for RNA interference (RNAi) in cells and larvae of the fall armyworm (FAW),Spodoptera frugiperda, a major worldwide pest. A highly efficient gene delivery formulation was prepared using a cationic biopolymer, PS, and a cationic lipid, Cellfectin (CF), complexed with dsRNA. The NPs were prepared by a two-step self-assembly method. The formation of NPs was revealed by dynamic light scattering and transmission electron microscopy. The formation of CF/dsRNA/PS NPs was spherical in shape and size, ranging from 20 to 100 nm with a positive charge (+23.3 mV). Interestingly, prepared CF/dsRNA/PS NPs could protect dsRNA (95%) from nuclease degradation and thus significantly improve the stability of dsRNA. Formulations prepared by combining EGFP DNA with CF/PS increased transfection efficiency in Sf9 cells compared to PS/EGFP and CF/EGFP NPs. Also, the PS/CF/dsRNA NPs enhanced the endosomal escape for the intracellular delivery of dsRNA. The gene knockdown efficiency was assessed in Sf9 Luciferase (Luc) stable cells after a 72 h incubation with CF/dsRNA/PS, PS/dsRNA, CF/dsRNA, or naked dsRNA. Knockdown of theLucgene was detected in CF/dsRNA/PS (76%) and PS/dsRNA (42.4%) not CF/dsRNA (19.5%) and naked dsRNA (10.3%) in Sf9 Luc cells. Moreover, CF/dsIAP/PS (25 μg of dsRNA targeting theinhibitor of apoptosis, IAP, gene of FAW) NPs showed knockdown of theIAPgene (39.5%) and mortality (55%) in FAW larvae. The insecticides that antagonize JH action and induce stoppage of feeding and precocious metamorphosis might work better to control these pests. Treating insects with JHA insecticides induces the expression of an early JH response gene, Krüppel homolog 1 (Kr-h1) by working through JH response elements (JHRE) present in its promoter. In this study, we identified JHREs present in the promoter of Kr-h1 gene of a global pest, S. frugiperda, and used them to develop a JHRE-reporter cell platform to screen for JH analogs. JHA, methoprene induced the expression of SfKr-h1 both in vitro and in vivo. JHRE present in the promoters of two SfKr-h1 isoforms, SfKr-h1α and SfKr-h1β were identified. In Sf9 cells, the knockout of isoform-specific JHRE affected JH response in an isoform-specific manner. We also found that S. frugiperda JHRE (SfJHRE) did not function in the mosquito Aedes aegypti Aag2 cells and Tribolium castaneum TcA cells. Similarly, Ae. aegypti AaJHRE and T. castaneum TcJHRE were only functional in cells derived from these insects. The nucleotide sequence at the 3'end to the conserved core JHRE E-box sequence seems to be responsible for the species specificity observed. Two stable cell lines expressing the luciferase and enhanced green fluorescent protein genes under the control of SfJHRE were established. These cell lines responded well to JHA; these two JHRE-reporter cell lines could be used in screening assays to identify insecticides to manage S. frugiperda and other major pests. Verson's glands are segmental pairs of dermal glands attached to the epidermis in lepidopteran larvae. They produce macromolecules during intermolt period and empty them during each molt. Morphological, histochemical, developmental, and protein analysis studies have been conducted to determine the functions of Verson's glands. However, the exact role of Verson's glands remains unclear. In our previous study, a strain of transgenic fall armyworm, Spdoptera frugiperda expressing green fluorescence protein (GFP) and Systemic RNA interference defective protein 1 (SID-1) from Caenorhabditis elegans was established to improve RNA interference (RNAi) efficiency. Unexpectedly, we found that GFP fluorescence was significantly brighter in Verson's glands than in other tissues. Also, RNAi efficiency improved more in Verson's glands than in other tissues. We took advantage of improved RNAi efficiency to explore the function of Verson's glands. RNA-seq analysis revealed that genes highly expressed in Verson's glands code for cuticular proteins, hemolymph proteins, molting fluid proteins, and antimicrobial peptides. Injection of dsRNA targeting essential genes interfered with Verson's gland growth. These studies revealed that Verson's glands contribute to hemolymph, cuticle, molting fluid, and immune response during molting. The enhanced green fluorescent protein and a red fluorescent protein, tdTomato genes driven by thehr5ie1promoter were found to be suitable for the identification of transgenic FAW. Multiple lines of transgenic FAW expressing Cas9 were generated and microinjection of sgRNAs into the embryos of these lines failed to induce target gene knockout. To overcome this problem, sgRNAs were expressed in FAW using U6-sgRNA and U6-tRNA-sgRNA systems,U6-tRNA-sgRNA system was found to be more efficient than U6-sgRNA system. Expression of Cas9 and sgRNAs in the same transgenic animal or in two separate strains followed by crossing them to bring Cas9 and sgRNA together resulted in an efficient knockout of target genes. The multiple transgenic CRISPR/Cas9-based genome editing methods developed provide invaluable tools for gene editing and functional genomics studies in this global pest and other lepidopteran pests. Recent genome and transcriptome sequencing efforts identified 117-425 P450 genes in the FAW, but their function in detoxifying plant toxins and insecticides is largely unknown. This study found that a P450 gene,SfCYP321A8, is upregulated in the first instar FAW larvae fed on deltamethrin. A transgenic FAW overexpressingSfCYP321A8was produced to investigate its function in deltamethrin resistance. Transgenic FAW expressing the gene coding for a P450 known to metabolize deltamethrin inTribolium castaneum,TcCYP6BQ9, was also produced. P450 genes are highly expressed in different tissues of transgenic larvae. The P450 activity in the midgut and fat body of both transgenic FAW lines is significantly higher than in wild-type larvae. Deltamethrin bioassays showed that the transgenic larvae expressingSfCYP321A8orTcCYP6BQ9are 10.3- or 15.3-fold more tolerant, respectively, than the wild-type larvae. These studies report on the production of FAW transgenic lines expressing P450 genes and show thatSfCYP321A8contributes to deltamethrin resistance in FAW. The transformation methods developed could be used in functional genomics studies in FAW and other lepidopteran pests. Promoter regions of 11 genes highly expressed in the midgut were identified and cloned. Baculoviruses expressing the luciferase gene under the control of these promoters were produced and tested in the FAW. These baculoviruses did not show significant luciferase activity in the FAW midgut. Four transgenic FAW lines expressing the luciferase gene under the control of the SfSP38/P2000, SfCalphotin/P2000, SfMG17/P2000, and SfCPH38/P2000 promoters were generated usingpiggyBac-based germline transformation methods. Significantly higher luciferase activity was detected in the midgut than in other tissues of transgenic FAW. The SfCPH38/P2000 promoter with the highest activity and midgut specificity was used to drive the expression of P450,SfCYP321A8,which is known to be involved in deltamethrin resistance. Higher mRNA levels ofSfCYP321A8and P450 activity were detected in the midgut of transgenic larvae than in wild-type larvae. Bioassays showed that transgenic larvae expressing SfCYP321A8 in the midgut are tolerant to deltamethrin. Here, we presented methods for the identification of midgut-specific promoters in the FAW and used them to study the role of P450 overexpression in the midgut on insecticide resistance.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Dhandapani RK, Gurusamy D, Palli SR. Protamine-Lipid-dsRNA Nanoparticles Improve RNAi Efficiency in the Fall Armyworm, Spodoptera frugiperda. J Agric Food Chem. 2022 Jun 8;70(22):6634-6643. doi: 10.1021/acs.jafc.2c00901. Epub 2022 May 25. PubMed PMID: 35612305.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Chen X, Palli SR. Identification of species-specific juvenile hormone response elements in the fall armyworm, Spodoptera frugiperda. Insect Biochem Mol Biol. 2022 Dec;151:103860. doi: 10.1016/j.ibmb.2022.103860. Epub 2022 Oct 27. PubMed PMID: 36374778.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Gene expression in Versons glands of the fall armyworm suggests their role in molting and immunity J Koo, X Chen, SR Palli Frontiers in Insect Science 3, 12
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Koo and Palli (2023) dsRNase1 contribution to dsRNA degradation activity in the Sf9 cells conditioned medium Front. Insect Sci., 24 January 2023 Sec. Insect Molecular Genetics Volume 3 - 2023 | https://doi.org/10.3389/finsc.2023.1118775
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Midgut-specific expression of CYP321A8 P450 gene increases deltamethrin tolerance in the fall armyworm Spodoptera frugiperda X Chen, SR Palli Journal of Pest Science, 1-13
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Transgenic overexpression of P450 genes confers deltamethrin resistance in the fall armyworm, Spodoptera frugiperda X Chen, SR Palli Journal of Pest Science 95 (3), 1197-1205
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Development of multiple transgenic CRISPR/Cas9 methods for genome editing in the fall armyworm, Spodoptera frugiperda X Chen, SR Palli Journal of Pest Science, 1-14

Progress 03/15/21 to 03/14/22

Outputs
Target Audience:Scientists working on Fall armyworm control Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A post-doctoral fellow was trained by this grant. How have the results been disseminated to communities of interest?Published three journal articles. What do you plan to do during the next reporting period to accomplish the goals?Studies on improving genome editing for use in functional genomics studies and impoving RNAi by knocking out dsRNases is in progress.

Impacts
What was accomplished under these goals? 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 inCaenorhabditis elegansrequires systemic RNA interference defective protein 1 (CeSid1). The expression of CeSid1in 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 expressedCeSid1in twoSpodoptera frugiperdacell lines, Sf9 and Sf17 cells derived from ovary and midgut, respectively. Expression ofCeSid1enhanced 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. TransgenicS. frugiperdaexpressingCeSid1were produced and tested for RNAi efficiency. RNAi efficiency enhancement due toCeSid1expression showed tissue specificity. Compared to RNAi efficiency in wild-typeS. frugiperda, CeSid1expressing transgenicS. frugiperdashowed 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. Thein vitrocell-type specific andin vivotissue-specific enhancement of RNAi efficiency byCeSid1inS. frugiperdaprovides valuable information for improving RNAi in insects such as those belonging to order Lepidoptera where RNAi is variable and inefficient.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Chen X, Palli SR. Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda. J Vis Exp. 2021 Sep 23;(175). doi: 10.3791/62714. PubMed PMID: 34633386.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Chen X, Koo J, Gurusamy D, Mogilicherla K, Reddy Palli S. Caenorhabditis elegans systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner. RNA Biol. 2021 Sep;18(9):1291-1299. doi: 10.1080/15476286.2020.1842632. Epub 2020 Nov 9. PubMed PMID: 33111632; PubMed Central PMCID: PMC8354605.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Palli SR. Epigenetic regulation of post-embryonic development. Curr Opin Insect Sci. 2021 Feb;43:63-69. doi: 10.1016/j.cois.2020.09.011. Epub 2020 Oct 14. Review. PubMed PMID: 33068783; PubMed Central PMCID: PMC8044252.

Progress 03/15/20 to 03/14/21

Outputs
Target Audience:Scientists working on Fall armyworm in USA and around the world. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student and one post-doctoral fellow were trained. How have the results been disseminated to communities of interest?Presented at conferences and published in a journal. What do you plan to do during the next reporting period to accomplish the goals?We will improve CRISPR/Cas9 methods for use in Fall armyworm.

Impacts
What was accomplished under these goals? 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 inCaenorhabditis elegansrequires systemic RNA interference defective protein 1 (CeSid1). The expression of CeSid1in 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 expressedCeSid1in twoSpodoptera frugiperdacell lines, Sf9 and Sf17 cells derived from ovary and midgut, respectively. Expression ofCeSid1enhanced 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. TransgenicS. frugiperdaexpressingCeSid1were produced and tested for RNAi efficiency. RNAi efficiency enhancement due toCeSid1expression showed tissue specificity. Compared to RNAi efficiency in wild-typeS. frugiperda, CeSid1expressing transgenicS. frugiperdashowed 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. Thein vitrocell-type specific andin vivotissue-specific enhancement of RNAi efficiency byCeSid1inS. frugiperdaprovides valuable information for improving RNAi in insects such as those belonging to order Lepidoptera where RNAi is variable and inefficient.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Chen, X., Koo, J., Gurusamy, D., Mogilicherla, K. & Reddy Palli, S. (2020) Caenorhabditis elegans systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner, RNA Biology. 1-9. doi.org/10.1080/15476286.2020.1842632.

Progress 03/15/19 to 03/14/20

Outputs
Target Audience:Scientists working on Fall armyworm Biology and management. Changes/Problems:Because of COVID-19 lockdown, access to the University of Kentucky laboratory facilities has been restricted, which resulted in slow down in the proposed research. What opportunities for training and professional development has the project provided?A post-doctoral fellow is beingtrained. How have the results been disseminated to communities of interest?Yes, the results have been presented at conferences and published in a journal. What do you plan to do during the next reporting period to accomplish the goals?We are working on using the CRISPR/Cas9 system developed to perform functional genomics to identify target genes for control of FAW.

Impacts
What was accomplished under these goals? The CRISPR/Cas9 system is an efficient genome editing method that can be used in functional genomics research. The fall armyworm, Spodoptera frugiperda, is a serious agricultural pest that has spread over most of the world. However, very little information is available on functional genomics for this insect. We performed CRISPR/Cas9-mediated site-specific mutagenesis of three target genes: two marker genes [Biogenesis of lysosome-related organelles complex 1 subunit 2 (BLOS2) and tryptophan 2, 3-dioxygenase (TO)], and a developmental gene, E93 (a key ecdysone-induced transcription factor that promotes adult development). The knockouts (KO) of BLOS2, TO and E93 induced translucent mosaic integument, olive eye color, and larval-pupal intermediate phenotypes, respectively. Sequencing RNA isolated from wild-type and E93 KO insects showed that E93 promotes adult development by influencing the expression of the genes coding for transcription factor, Krüppel homolog 1, the pupal specifier, Broad-Complex, serine proteases, and heat shock proteins. Often, gene-edited insects display mosaicism in which only a fraction of the cells are edited as intended, and establishing a homozygous line is both costly and time-consuming. To overcome these limitations, a method to completely KO the target gene in S. frugiperda by injecting the Cas9 protein and multiple sgRNAs targeting one exon of the E93 gene into embryos was developed. Ten percent of the G0 larvae exhibited larval-pupal intermediates. The mutations were confirmed by T7E1 assay, and the mutation frequency was determined as >80%. Complete KO of the E93 gene was achieved in one generation using the multiple sgRNA method, demonstrating a powerful approach to improve genome editing in lepidopteran and other non-model insects.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhu GH, Chereddy SCRR, Howell JL, Palli SR. Genome editing in the fall armyworm, Spodoptera frugiperda: Multiple sgRNA/Cas9 method for identification of knockouts in one generation. Insect Biochem Mol Biol. 2020 Apr 7;122:103373. doi: 10.1016/j.ibmb.2020.103373. [Epub ahead of print] PubMed PMID: 32276113.