Harnessing the highly adaptable barnase-barstar system for genetic biocontrol of Aedes aegypti

TY - JOUR

T1 - Harnessing the highly adaptable barnase-barstar system for genetic biocontrol of Aedes aegypti

AU - Nevard, Katherine

AU - Ang Xin De, Joshua

AU - Anderson, Michelle

AU - Gonzalez Fernandez, Estela

AU - Shackleford, Lewis

AU - Alphey, Luke Stephen

N1 - © The Author(s) 2025

PY - 2025/8/4

Y1 - 2025/8/4

N2 - Toxin-antidote gene drive systems are a class of vector control methods which mainly aim to modify a wild population into one with refractoriness to pathogens. However, while toxins are aplenty, antidotes which fully nullify the effects of the toxins are rare. Here we describe the adaptation of a toxin-antidote pair, barnase-barstar, respectively, to Aedes aegypti, the vector of dengue, Zika, yellow fever, and chikungunya viruses. The pair was originally identified in Bacillus amyloliquefaciens where barnase acts as an extracellular ribonuclease and barstar binds to it, thereby inhibiting its intracellular enzymatic activity. For synthetic biology, this effector pair is highly attractive due to their small sizes: 110 and 89 amino acid residues for barnase and barstar, respectively and high binding affinity.Firstly, we show that barnase and barstar expressed from plasmids can cause lethality and rescue, respectively, in Aag2 cells and thus we proceeded to generate transgenic strains expressing either TRE-Barnase-PUb-Barstar (inactive toxin and ubiquitous antidote) or TRE-barnase (inactive toxin). In the absence of barstar, ubiquitous expression of barnase using trunPUb-tTAV resulted in up to 100% lethality. AeAct4-tTAV (female indirect flight muscle) and AeCPA-tTAV (female midgut post bloodfeeding) activation of barnase caused 100% of females to be flightless and reduced female fertility from an average of 70.7% to 5.1% (Kruskal-Wallis test, n=71, p<0.0001), respectively. This rescue was only partial for AeAct4-tTAV with 37% of females carrying both TRE-Barnase-PUb-Barstar and AeAct4-tTAV were able to fly, likely due to the lack of an overlap of expression between PUb and AeAct4. In conclusion, we have shown barnase-barstar to be a highly adaptable toxin-antidote pair which is now added to the panel of available effectors to establish toxin-antidote gene drive systems.

AB - Toxin-antidote gene drive systems are a class of vector control methods which mainly aim to modify a wild population into one with refractoriness to pathogens. However, while toxins are aplenty, antidotes which fully nullify the effects of the toxins are rare. Here we describe the adaptation of a toxin-antidote pair, barnase-barstar, respectively, to Aedes aegypti, the vector of dengue, Zika, yellow fever, and chikungunya viruses. The pair was originally identified in Bacillus amyloliquefaciens where barnase acts as an extracellular ribonuclease and barstar binds to it, thereby inhibiting its intracellular enzymatic activity. For synthetic biology, this effector pair is highly attractive due to their small sizes: 110 and 89 amino acid residues for barnase and barstar, respectively and high binding affinity.Firstly, we show that barnase and barstar expressed from plasmids can cause lethality and rescue, respectively, in Aag2 cells and thus we proceeded to generate transgenic strains expressing either TRE-Barnase-PUb-Barstar (inactive toxin and ubiquitous antidote) or TRE-barnase (inactive toxin). In the absence of barstar, ubiquitous expression of barnase using trunPUb-tTAV resulted in up to 100% lethality. AeAct4-tTAV (female indirect flight muscle) and AeCPA-tTAV (female midgut post bloodfeeding) activation of barnase caused 100% of females to be flightless and reduced female fertility from an average of 70.7% to 5.1% (Kruskal-Wallis test, n=71, p<0.0001), respectively. This rescue was only partial for AeAct4-tTAV with 37% of females carrying both TRE-Barnase-PUb-Barstar and AeAct4-tTAV were able to fly, likely due to the lack of an overlap of expression between PUb and AeAct4. In conclusion, we have shown barnase-barstar to be a highly adaptable toxin-antidote pair which is now added to the panel of available effectors to establish toxin-antidote gene drive systems.

U2 - 10.1038/s42003-025-08588-6

DO - 10.1038/s42003-025-08588-6

M3 - Article

SN - 2399-3642

VL - 8

JO - Communication Biology

JF - Communication Biology

M1 - 1154

ER -