Efficient RNA Virus Targeting via CRISPR/CasRx in Fish.

Area of Science:

• Molecular Biology
• Virology
• Gene Editing Technology

Background:

• RNA viruses pose significant threats to vertebrates and mammals, exhibiting rapid evolution that complicates antiviral strategies.
• The CRISPR/Cas13 system offers a targeted approach to RNA modification, with CasRx (Cas13d) showing potential for RNA virus interference.
• Nervous necrosis virus (NNV) is a major RNA virus pathogen in aquaculture, causing substantial economic losses globally.

Purpose of the Study:

• To engineer and validate a highly effective CasRx system for interfering with RNA viruses in fish.
• To assess the in vitro and in vivo efficacy of CasRx against the red-spotted grouper nervous necrosis virus (RGNNV).
• To establish CasRx as a potential tool for RNA-guided immunity against RNA viruses in vertebrates.

Main Methods:

• Designed synthetic messenger RNA (mRNA) encoding the CasRx effector.
• Utilized CRISPR RNAs (crRNAs) to guide CasRx to target RGNNV RNA.
• Evaluated interference efficiency in both cell cultures (in vitro) and live fish (in vivo models).

Main Results:

• Successfully engineered a robust CasRx system for potent RNA virus interference in fish.
• Demonstrated significant inhibition of RGNNV infections using the CasRx system in vitro and in vivo.
• Confirmed CasRx's effectiveness and suitability for studying RNA virus biology in fish models.

Conclusions:

• The CRISPR/CasRx system provides a novel and effective mechanism for engineering RNA-guided immunity against RNA viruses in fish.
• This technology holds potential for developing new antiviral strategies against various RNA viruses in vertebrates.
• CasRx is a promising tool for future research into RNA virus pathogenesis and control in aquaculture and beyond.