A New Method for Programmable RNA Editing Using CRISPR Effector Cas13X.1.

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• RNA Biology

Background:

• Type VI CRISPR-Cas13 systems are unique RNA-targeting gene editing tools.
• Existing CRISPR-Cas13 systems face delivery challenges for in vivo applications.
• Cas13X.1 is a newly discovered, smaller Cas13 protein with potential for improved delivery.

Purpose of the Study:

• To develop and evaluate a CRISPR-Cas13X.1-based RNA editing system in mammalian cells.
• To assess the efficacy and safety of Cas13X.1 for targeting both exogenous and endogenous genes.
• To explore the potential of Cas13X.1 for in vivo delivery and therapeutic applications, particularly in cancer.

Main Methods:

• Constructed recombinant plasmids using the Cas13X.1 backbone to target mCherry and transketolase (TKT) genes.
• Evaluated RNA knockdown efficiency, protein expression levels, and downstream gene transcript levels.
• Assessed cellular toxicity by examining cell morphology, RNA integrity, and apoptosis.

Main Results:

• Cas13X.1 system achieved significant degradation of TKT and mCherry transcripts, with up to 50% knockdown efficiency.
• Downregulation of target transcripts led to reduced corresponding protein levels.
• Synchronous downregulation of TKT-related metabolic enzymes indicated pathway disruption.
• The Cas13X.1 system demonstrated low cellular toxicity and did not affect cell morphology, RNA integrity, or apoptosis.

Conclusions:

• The developed Cas13X.1 system exhibits potent RNA knockdown capabilities in mammalian cells with minimal toxicity.
• Its smaller molecular weight offers advantages for in vivo delivery compared to other CRISPR-Cas13 systems.
• Targeting TKT transcripts with Cas13X.1 presents a potential strategy for anti-cancer therapy research.