Comparison of CRISPR-Cas13b RNA base editing approaches for USH2A-associated inherited retinal degeneration

Area of Science:

• Molecular Biology
• Gene Therapy
• Ophthalmology

Background:

• CRISPR-Cas13 systems offer potential for precise RNA point mutation correction.
• Adenosine deaminase acting on RNA (ADAR) effectors enable targeted A-to-I base conversions.
• USH2A gene mutations are a common cause of inherited retinal disease and Usher syndrome.

Purpose of the Study:

• To compare the efficacy of Cas13 effectors (PspCas13b and Cas13bt3) for repairing the USH2A gene mutation.
• To develop and characterize a mouse model for evaluating base editors in inherited retinal disease.
• To assess the therapeutic potential of transcriptome-targeting gene editing for retinal diseases.

Main Methods:

• Comparison of PspCas13b and Cas13bt3 effectors for USH2A gene repair in cultured cells using various guide RNAs (gRNAs) and promoters.
• Development and characterization of a Usher syndrome mouse model with a specific USH2A mutation (c.11840 G>A).
• In vivo evaluation of Cas13 effectors delivered via Adeno-Associated Virus (AAV) in mouse photoreceptors.

Main Results:

• Up to 80% efficiency in repairing the USH2A mutation (c.11864 G>A and c.11840 G>A) in cultured cells.
• In vivo RNA editing rates in photoreceptors ranged from 0.32% to 2.04%, with PspCas13b showing greater efficiency than Cas13bt3.
• Restoration and correct localization of usherin protein in the connecting cilium of mice treated with PspCas13b constructs.

Conclusions:

• PspCas13b and Cas13bt3 are effective Cas13 effectors for correcting the USH2A mutation at the RNA level.
• The developed mouse model is suitable for evaluating base editors for inherited retinal diseases.
• Transcriptome-targeting gene editing holds promise for treating retinal diseases like Usher syndrome.