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Development of multiple transgenic CRISPR/Cas9 methods for genome editing in the fall armyworm, Spodoptera frugiperda

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Abstract

The use of CRISPR/Cas9 system in model insects has facilitated functional genomics studies. However, this system has not been applied to many pest insects. Here, we report on the establishment of multiple transgenic CRISPR/Cas9-based genome editing methods in a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda. To identify fluorescent proteins suitable for screening for transgenic FAW, nine transgenic lines expressing genes coding for fluorescent proteins under the control of different promoters were produced and evaluated. The enhanced green fluorescent protein and a red fluorescent protein, tdTomato genes driven by the hr5ie1 promoter 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.

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References

  • Abdelgaffar H, Perera OP, Jurat‐Fuentes JL (2021) ABC transporter mutations in C ry 1F‐resistant fall armyworm (Spodoptera frugiperda) do not result in altered susceptibility to selected small molecule pesticides Pest Manage Sci 77:949–955

  • Chaverra-Rodriguez D et al. (2018) Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing. Nat Commun 9:3008. Doi:https://doi.org/10.1038/s41467-018-05425-9

  • Chen X, Chereddy SC, Gurusamy D, Palli SR (2020a) Identification and characterization of highly active promoters from the fall armyworm, Spodoptera Frugiperda. Insect Biochem Mol Biol 126:103455

    Article  CAS  PubMed  Google Scholar 

  • Chen X, Koo J, Gurusamy D, Mogilicherla K, Reddy Palli S (2020b) 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, pp 1–9. Doi:https://doi.org/10.1080/15476286.2020b.1842632

  • Chen X, Palli SR (2021) Hyperactive piggyBac transposase-mediated germline transformation in the fall armyworm, Spodoptera frugiperda. J Vis Exp, p e62714. Doi:https://doi.org/10.3791/62714

  • Chen X, Tan A, Palli SR (2020c) Identification and functional analysis of promoters of heat-shock genes from the fall armyworm, Spodoptera Frugiperda. Sci Rep 10:1–9

    Google Scholar 

  • Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Day RN, Davidson MW (2009) The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38:2887–2921

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dong Z et al. (2018) Establishment of a baculovirus-inducible CRISPR/Cas9 system for antiviral research in transgenic silkworms. Appl Microbiol Biotechnol 102:9255–9265

  • Dong Z et al. (2019) Construction and application of an HSP70 promoter-inducible genome editing system in transgenic silkworm to induce resistance to Nosema bombycis. Appl Microbiol Biotechnol 103:9583–9592

  • Ferreira R, Skrekas C, Nielsen J, David F (2018) Multiplexed CRISPR/Cas9 genome editing and gene regulation using Csy4 in saccharomyces cerevisiae. ACS Synth Biol 7:10–15. Doi:https://doi.org/10.1021/acssynbio.7b00259

  • Gao Y, Zhao Y (2014) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing Journal of Integrated. Plant Biol 56:343–349. https://doi.org/10.1111/jipb.12152

    Article  CAS  Google Scholar 

  • Gouin A et al. (2017) Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges. Sci Rep 7:1–12

  • Gregory M, Alphey L, Morrison NI, Shimeld SM (2016) Insect transformation with piggyBac: getting the number of injections just right. Insect Mol Biol 25:259–271. https://doi.org/10.1111/imb.12220

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gui F et al. (2020) Genomic and transcriptomic analysis unveils population evolution and development of pesticide resistance in fall armyworm Spodoptera frugiperda. Protein Cell, pp 1–19

  • Han W, Tang F, Zhong Y, Zhang J, Liu Z (2021) Identification of yellow gene family and functional analysis of Spodoptera frugiperda yellow-y by CRISPR/Cas9. Pesticide Biochem Physiol 178:104937

  • Heu CC, McCullough FM, Luan J, Rasgon JL (2020) CRISPR-Cas9-based genome editing in the silverleaf whitefly (Bemisia tabaci). CRISPR J 3:89–96

  • Hillary VE, Ceasar SA, Ignacimuthu S (2020) Genome engineering in insects: focus on the CRISPR/Cas9 system. In: Genome engineering via CRISPR-Cas9 System. Elsevier, pp 219–249

  • Horn C, Schmid BG, Pogoda FS, Wimmer EA (2002) Fluorescent transformation markers for insect transgenesis. Insect Biochem Mol Biol 32:1221–1235

    Article  CAS  PubMed  Google Scholar 

  • Huang Y et al. (2017) Functional characterization of Pol III U6 promoters for gene knockdown and knockout in Plutella xylostella. Insect Biochem Mol Biol 89:71–78. Doi:https://doi.org/10.1016/j.ibmb.2017.08.009

  • Jin M-h, Tao J-h, qi L, Cheng Y, Sun X-x, Wu K-m, Xiao Y-T (2021) Genome editing of the SfABCC2 gene confers resistance to Cry1F toxin from Bacillus thuringiensis in Spodoptera frugiperda. J Integrat Agric 20:815–820

  • Kakumani PK, Malhotra P, Mukherjee SK, Bhatnagar RK (2014) A draft genome assembly of the army worm Spodoptera Frugiperda. Genomics 104:134–143

    Article  CAS  PubMed  Google Scholar 

  • Kandul NP et al (2021) Genetically encoded CRISPR components yield efficient gene editing in the invasive pest drosophila Suzukii. CCRISPR J 4:739–751. https://doi.org/10.1089/crispr.2021.0032

    Article  CAS  Google Scholar 

  • Li J-J, Shi Y, Wu J-N, Li H, Smagghe G, Liu T-X (2021) CRISPR/Cas9 in Lepidopteran insects: progress, application and prospects. J Insect Physiol, pp 104325

  • Li M, Bui M, Yang T, Bowman CS, White BJ, Akbari OS (2017) Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector Aedes Aegypti. Proc Nat Acad Sci 114:E10540–E10549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li M et al (2020) Development of a confinable gene drive system in the human disease vector Aedes Aegypti. Elife 9:e51701

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li Z et al (2015) Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori. Proc R Soc b Biol Sci 282:20150513

    Article  Google Scholar 

  • Lin C-C, Potter CJ (2016) Non-Mendelian dominant maternal effects caused by CRISPR/Cas9 transgenic components in Drosophila melanogaster G3: Genes. Genomes Genet 6:3685–3691

    CAS  Google Scholar 

  • Liu X-L et al. (2020) The molecular basis of host selection in a crucifer-specialized moth. Current Biol 30:4476–4482

  • Lu K, Song Y, Zeng R (2020) The role of cytochrome P450-mediated detoxification in insect adaptation to xenobiotics. Current Opin Insect Sci 43:103–107. Doi: https://doi.org/10.1016/j.cois.2020.11.004.

  • Ma H, Wu Y, Dang Y, Choi J-G, Zhang J, Wu H (2014) Pol III promoters to express small RNAs: delineation of transcription initiation. Mol Therapy Nucleic Acids 3:e161

    Article  CAS  Google Scholar 

  • Mabashi-Asazuma H, Jarvis DL (2017) CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus–insect cell system. Proc Nat Acad Sci 114:9068–9073

  • Martins S et al. (2012) Germline transformation of the diamondback moth, Plutella xylostella L., using the piggyBac transposable element. Insect Mol Biol 21:414–421

  • Montezano DG et al. (2018) Host plants of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas. Afr Entomol 26:286–300

  • Nagoshi RN et al (2017) Fall armyworm migration across the Lesser Antilles and the potential for genetic exchanges between North and South American populations. PLoS ONE 12:e0171743

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Naik NG, Lo Y-W, Wu T-Y, Lin C-C, Kuo S-C, Chao Y-C (2018) Baculovirus as an efficient vector for gene delivery into mosquitoes. Sci Rep 8:1–14

    Article  Google Scholar 

  • Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK (2014) Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell 54:698–710. https://doi.org/10.1016/j.molcel.2014.04.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Port F, Bullock SL (2016) Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat Methods 13:852–854

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Port F, Muschalik N, Bullock SL (2015) Systematic evaluation of Drosophila CRISPR tools reveals safe and robust alternatives to autonomous gene drives in basic research G3: Genes. Genomes Genet 5:1493–1502

    CAS  Google Scholar 

  • Rangan P, DeGennaro M, Lehmann R (2008) Regulating gene expression in the Drosophila germ line. Cold Spring Harbor symposia on quantitative biology. Cold Spring Harbor Laboratory Press, pp 1–8

    Google Scholar 

  • Rylee JC, Nin-Velez A, Mahato S, Helms KJ, Wade MJ, Zentner GE, Zelhof AC (2021) Generating and testing the efficacy of transgenic Cas9 in Tribolium castaneum. Doi:https://doi.org/10.1101/2021.10.28.466351

  • Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes Nat Biotechnol 32:347–355

  • Shemiakina I et al (2012) A monomeric red fluorescent protein with low cytotoxicity. Nat Commun 3:1–7

    Article  Google Scholar 

  • Taning CNT, Van Eynde B, Yu N, Ma S, Smagghe G (2017) CRISPR/Cas9 in insects: applications, best practices and biosafety concerns. J Insect Physiol 98:245–257

  • Tepa-Yotto GT et al. (2021) Global habitat suitability of Spodoptera frugiperda (JE Smith)(Lepidoptera, Noctuidae): key parasitoids considered for its biological control. Insects 12:273

  • Thyme SB, Akhmetova L, Montague TG, Valen E, Schier AF (2016) Internal guide RNA interactions interfere with Cas9-mediated cleavage. Nat Commun 7:1–7

  • Wu K, Shirk PD, Taylor CE, Furlong RB, Shirk BD, Pinheiro DH, Siegfried BD (2018) CRISPR/Cas9 mediated knockout of the abdominal-A homeotic gene in fall armyworm moth (Spodoptera frugiperda). PloS One 13:e0208647

  • Xiao H et al (2020) The genetic adaptations of fall armyworm Spodoptera frugiperda facilitated its rapid global dispersal and invasion. Mol Ecol Resour 20:1050–1068

    Article  CAS  PubMed  Google Scholar 

  • Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Nat Acad Sci 112:3570–3575

  • Xu J et al (2019a) Identification of a Germline-expression promoter for genome editing in Bombyx Mori. Insect Sci 26:991–999

    Article  CAS  PubMed  Google Scholar 

  • Xu J, Xu X, Zhan S, Huang Y (2019b) Genome Editing in Insects: Current Status and Challenges. Nat Sci Rev 6:399–401

    Article  CAS  Google Scholar 

  • Xu X et al. (2021) Towards CRISPR/Cas9-based gene drive in the diamondback moth Plutella xylostella. Doi:https://doi.org/10.1101/2021.10.05.462963

  • Zeng B et al (2016) Expansion of CRISPR targeting sites in Bombyx mori. Insect Biochem Mol Biol 72:31–40

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Wang J, Wang Z, Zhang Y, Shi S, Nielsen J, Liu Z (2019) A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat Commun 10:1–10

  • Zhou J, Lin J, Zhou C, Deng X, Xia B (2011) Cytotoxicity of red fluorescent protein DsRed is associated with the suppression of Bcl-xL translation. FEBS Lett 585:821–827

    Article  CAS  PubMed  Google Scholar 

  • Zhu G-H, Chereddy SC, Howell JL, Palli SR (2020) Genome editing in the fall armyworm, Spodoptera frugiperda: multiple sgRNA/Cas9 method for identification of knockouts in one generation. Insect Biochem Mol Biol 122:103373

  • Zhu G-H, Jiao Y, Chereddy SC, Noh MY, Palli SR (2019) Knockout of juvenile hormone receptor, Methoprene-tolerant, induces black larval phenotype in the yellow fever mosquito, Aedes aegypti. Proc Natl Acad Sci 116:21501–21507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu G-H et al. (2016) Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing. Insect Biochem Mol Biol 75:1–9

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Acknowledgements

We thank Jeff Howell from the University of Kentucky for help with FAW rearing. The research is supported by Agriculture and Food Research Initiative Competitive Grant No. 2019-67013-29351 and the National Institute of Food and Agriculture, US Department of Agriculture (2353057000).

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  1. Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, USA

    Xien Chen & Subba Reddy Palli

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  1. Xien Chen
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  2. Subba Reddy Palli
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XC and SRP designed and conceived the study. XC performed experiments. XC and SRP analyzed data and wrote the manuscript.

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Correspondence to Subba Reddy Palli.

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Communicated by Su Wang.

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Chen, X., Palli, S.R. Development of multiple transgenic CRISPR/Cas9 methods for genome editing in the fall armyworm, Spodoptera frugiperda. J Pest Sci 96, 1637–1650 (2023). https://doi.org/10.1007/s10340-022-01546-9

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