Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Alternative splicing and other mechanisms generate diverse RNA and protein isoforms, crucial for cellular functions.
• Characterizing isoform function is challenging due to limited methods for specific expression modulation and complex data analysis.

Purpose of the Study:

• To develop and validate an efficient, isoform-specific RNA knockdown strategy.
• To provide computational tools supporting experimental design and analysis for isoform studies.

Main Methods:

• Developed an RNA-targeting CRISPR/Cas13d system utilizing guide RNAs spanning exon-exon junctions.
• Tested the strategy across various genes, alternative splicing events, and cell types.
• Created accompanying computational tools for experimental design and data analysis.

Main Results:

• Demonstrated robust and isoform-specific RNA knockdown using the junction-targeting CRISPR/Cas13d system.
• Validated the method's effectiveness across diverse alternative splicing events and cell lines.
• Showcased the utility of the developed computational tools for experimental workflows.

Conclusions:

• The developed junction-targeting CRISPR/Cas13d system provides a powerful tool for isoform-specific gene knockdown.
• This method overcomes previous limitations in studying alternative isoform function.
• Enables precise investigation of the roles of distinct RNA and protein isoforms in cellular processes.