High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• RNA Therapeutics

Background:

• CRISPR-Cas13 systems enable targeted RNA degradation.
• Collateral degradation of bystander RNAs limits in vivo applications of Cas13.
• Need for Cas13 variants with reduced off-target effects.

Purpose of the Study:

• Develop a reporter system to detect collateral effects of Cas13.
• Screen engineered Cas13 variants for high-fidelity RNA degradation.
• Evaluate safety and efficacy of high-fidelity Cas13 variants in vitro and in vivo.

Main Methods:

• Designed a dual-fluorescence reporter system for screening Cas13 variants in mammalian cells.
• Engineered and screened over 200 Cas13 variants.
• Assessed on-target activity, collateral activity, transcriptome-wide off-targets, and cell growth effects.
• Validated high-fidelity variants in transgenic mice and AAV-mediated somatic cell targeting.

Main Results:

• Identified several high-fidelity Cas13 variants (e.g., Cas13d, Cas13X) with efficient on-target RNA degradation.
• These variants exhibited markedly reduced collateral activity compared to wild-type Cas13.
• Transcriptome-wide off-targets and cell growth arrest were absent for high-fidelity variants.
• No detectable collateral damage was observed in vivo (transgenic mice, somatic cell targeting).

Conclusions:

• High-fidelity Cas13 variants offer precise RNA degradation with minimal collateral effects.
• These engineered variants overcome limitations of wild-type Cas13 for in vivo applications.
• Available high-fidelity Cas13 variants are suitable for basic research and therapeutic RNA degradation.