Team:CAU China/Design

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We design a locust bio-pesticide by applying with RNAi technology targeting to different stage of locust in order to inhibit the growing rate of locust. We employ the synthetic biology method to Saccharomyce cerevisiae and Metarhizium acridum which can affect locust by interfering critical genes in locust metabolism and growth.

• RNAi techonolgy
  RNAi is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules. During the process, double strand RNA would be cleaved into siRNA by DICER. siRNA will assemble with multiple proteins and form RNA-induced silencing complex (RISC). RNAi can specifically identify target gene and suppress its expression. This technology is precise, efficient and stable. Before we designed dsRNA sequence, we first blast the target gene to human, crops and other species to prevent the affect of RNAi to them. We chose sequence which has low-identity to those species so that our dsRNA wouldn’t affect to them. Before we started our experiment, we searched and read huge amount of papers about RNAi design on locust. Finally, we chose CHS (chitin synthase), TSP (trypsin-like serine protease) and ATPase (vacuolar ATPase subunit H) gene as our target gene. By using specific program, we attain our dsRNA sequence and then we decided to measure its effect by using transferred yeast and metarhizium.

  Phase of Locust	Target gene	Mortality
  Ⅲ instar	LmHR3	100%
  Ⅳ instar	Lm-TSP	100%
  	LmHR3	100%
  Ⅴ instar	LmSPC1	86.7%
  	Lm-TSP	46%
  	LmHR3	100%
  	Lm-Vhasfd	96.7%
  	LmE75	100%
  Adult	LmSPC1	80%

  Before we started our experiment, we searched and read huge amount of papers about RNAi design on locust. Finally, we chose CHS (chitin synthase), TSP (trypsin-like serine protease) and ATPase (vacuolar ATPase subunit H) gene as our target gene.
  By using specific program, we attain our dsRNA sequence and then we decided to measure its effect by using transferred yeast and metarhizium.
• CarH Light-Control system
  Light control system has many advantages comparing to chemical induction.
  1.fast and efficient rate of inducing
  2.easy to manipulate by computer
  3.low toxicity to cell
  To select an appropriate light control system for our project, there are three factors we should consider:
  1.showing high induction
  2.no extra chemical reagent
  3.light has no influence to strain
  Consequently, we choose CarH light control system.

  Light control system	Light	organism	Induction fold	othre
  Phy/PIF3interactio11	R/FR	Yeast	1000	Exogenously added PCB
  CRY2/CIB1 with TALE DNA operon12	blue	mammal	Up to 30	TALE DNA operon should be constructed
  CRY2/CIB1 and CRISPR/Cas9system10	blue	mammal	500
  UVR8/COP1interaction5	UVB	mammal	800
  CarH system2	Green	Mammal and plant	350
  EL2226	blue	E.coli and yeast	＜5
  OptoINVRT and OptoEXP6	blue	yeast	＜5
  Yeast optogenetic control system3	blue	yeast	3

  CarH protein is from Thermus thermophilus. This protein has cobalamine (Vitamin B12), which has a photosensitive Co-C bond, as a prosthetic group. In the dark, CarH is a tetramer that binds strongly to CarO DNA operon and represses downstream protein translation². When excited by green light, the photosensitive Co-C bond breaks, this disrupts the tetramer structure, and then gene expression can start. From the research we know CarH maximum absorption spectrum is 525nm, we decided to apply green light control system to manipulate yeast dsRNA synthesis. When the yeast is incubated in dark condition, CarH will bind to dsRNA synthesis part which will block the dsRNA synthesis. When exposing to 525nm light environment, CarH tetramer photolysis which resulted in dsRNA synthesis expression.
• dsRNA measurement
  siRNA is unstable in cell and could only function several days. We decided to employ dsRNA in our experiment. dsRNA is composed of an antisense and sense RNA sequence of targeted gene and a single strand RNA loop structure. dsRNA would be combined to plasmid and deliver into host cell, converted into siRNA and finally interfered gene expression. To measure the concentration of dsRNA, we decided to use pepper RNA. Pepper RNA is a fluorescent RNA, an aptamer that binds and activate fluorescent dyes. Pepper RNA can bind to (4-((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenylacetonitrile (HBC) and emit stable fluorescence⁸. We then measure the concentration of dsRNA by fluorescence strength and compare its value to standard curve.
  
• 
  
  Mechanism of Metarhizium insecticides
  Metarhizium has a high rate of pathogenic to 3rd instar locust and 4th, about mortality rate from 70%~80%. On the contrast, adult locust has a related low mortality of 60%¹³.
  The process of Metarhizium virulence can be divided into three steps:
  1.spore attachment：penetration through the body wall；
  2.colonization in vivo： evade the immune system of insect hosts；
  3.killing the host：produce compounds such as beauvericin and destruxin and re-sporulation.
  
  In order to enhance Metarhizium virulence ability to locust, we employ RNAi technics, which shows that high infection to Metarhizium when transformed dsRNA sequence targeted to locust gene.
• References
  1.Niu, J. et al., Beyond insects: current status and achievements of RNA interference in mite pests and future perspectives. PEST MANAG SCI 74 2680 (2018).
  2.Chatelle, C. et al., A Green-Light-Responsive System for the Control of Transgene Expression in Mammalian and Plant Cells. ACS SYNTH BIOL 7 1349 (2018).
  3.Pathak, G. P., Strickland, D., Vrana, J. D. & Tucker, C. L., Benchmarking of Optical Dimerizer Systems. ACS SYNTH BIOL 3 832 (2014).
  4. Zhang, W. & Xia, Y., ER type I signal peptidase subunit (LmSPC1) is essential for the survival of Locusta migratoria manilensis and affects moulting, feeding, reproduction and embryonic development. INSECT MOL BIOL 23 269 (2014).
  5.Müller, K. et al., Multi-chromatic control of mammalian gene expression and signaling. NUCLEIC ACIDS RES 41 e124 (2013).
  6. Zhao, E. M. et al., Optogenetic regulation of engineered cellular metabolism for microbial chemical production. NATURE 555 683 (2018).
  7.Zotti, M. et al., RNA interference technology in crop protection against arthropod pests, pathogens and nematodes. PEST MANAG SCI 74 1239 (2018).
  8. Chen, X. et al., Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. NAT BIOTECHNOL 37 1287 (2019).
  9. 农向群 et al., 真菌防治蝗虫研究进展. 中国生物防治学报 1 (2020).
  10. Polstein, L. R. & Gersbach, C. A., A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. NAT CHEM BIOL 11 198 (2015).
  11.Shimizu-Sato, S., Huq, E., Tepperman, J. M. & Quail, P. H., A light-switchable gene promoter system. NAT BIOTECHNOL 20 1041 (2002).
  12. Konermann, S. et al., Optical control of mammalian endogenous transcription and epigenetic states. NATURE 500 472 (2013).
  13.刘银民, 甘肃农业大学, 2017.
  14.胡军, 重庆大学, 2015.

1. RNAi techonolgy
2. CarH Light-Control system
3. dsRNA measurement
4. Mechanism
5. References