Enhanced RNA-targeting CRISPR-Cas technology in zebrafish

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas13 systems offer RNA targeting but face challenges with collateral activity and in vivo efficiency.
• Optimizing delivery and guide RNA (gRNA) design is crucial for enhancing CRISPR-Cas13 applications in live organisms.

Purpose of the Study:

• To optimize transient CRISPR-RfxCas13d formulations for enhanced RNA targeting in zebrafish.
• To evaluate the efficacy and specificity of modified gRNAs and computational models for predicting gRNA activity in vivo.
• To explore alternative RNA-targeting CRISPR-Cas systems for zebrafish applications.

Main Methods:

• Chemically modified gRNAs were employed to improve loss-of-function phenotypes.
• Nuclear RNA targeting was enhanced through optimized transient formulations (ribonucleoprotein complexes or mRNA-gRNA).
• Computational models were compared to identify the most accurate for predicting in vivo gRNA activity.

Main Results:

• Transient CRISPR-RfxCas13d effectively depleted endogenous mRNAs in zebrafish embryos.
• Collateral effects were minimal, except when targeting highly abundant or ectopic RNAs.
• Optimized formulations and chemically modified gRNAs improved system competence in vivo.

Conclusions:

• Transient CRISPR-Cas13d systems can be effectively optimized for RNA targeting in zebrafish embryos.
• This study provides a framework for advancing in vivo RNA targeting applications using CRISPR-Cas technologies.
• Alternative systems like CRISPR-Cas7-11 and CRISPR-DjCas13d were successfully implemented, broadening RNA targeting options.