Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors.

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Class 2 CRISPR-Cas systems provide adaptive immunity in microbes.
• Previous research focused on DNA-targeting systems, leaving RNA-targeting systems less explored.

Purpose of the Study:

• To identify and characterize novel RNA-targeting CRISPR-Cas systems.
• To engineer a novel RNA-targeting enzyme for potential therapeutic applications.

Main Methods:

• Bioinformatic analysis of prokaryotic genomes and metagenomes.
• Biochemical characterization and protein engineering of identified CRISPR-Cas orthologs.
• In vitro and in vivo functional assays in human cells and neuronal models.

Main Results:

• Identification and classification of a new RNA-targeting CRISPR-Cas system, type VI-D.
• Engineering of a compact, highly efficient RNA-targeting ribonuclease, CasRx, from Ruminococcus flavefaciens XPD3002.
• Demonstration of CasRx-mediated RNA knockdown with high efficiency and specificity in human cells.
• Application of catalytically inactive CasRx to manipulate alternative splicing and alleviate tau dysregulation in a frontotemporal dementia model.

Conclusions:

• CasRx represents a novel, programmable RNA-binding module for transcriptome engineering.
• The type VI-D CRISPR-Cas system and its CasRx effector offer a versatile platform for RNA targeting.
• CasRx holds promise for future therapeutic development, particularly in genetic and neurodegenerative diseases.