CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery.

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Genetic Engineering

Background:

• Existing genome editing tools often require double-strand breaks (DSBs) or DNA templates, limiting their application for large gene delivery.
• There is a need for advanced genome editing systems capable of precise, template-free insertion of large genetic payloads.

Purpose of the Study:

• To introduce and characterize CRISPR-Enabled Autonomous Transposable Element (CREATE), a novel RNA-based genome editing system.
• To demonstrate the capability of CREATE for targeted insertion of large gene expression cassettes into specific genomic loci.

Main Methods:

• Utilized a modified LINE-1 (L1) mRNA to encapsulate a payload gene for delivery.
• Employed a CRISPR/Cas9 nickase to guide L1-mediated reverse transcription and integration at targeted genomic sites.
• Validated CREATE system in human cell lines and primary T cells, assessing insertion specificity and off-target effects.

Main Results:

• Successfully demonstrated programmable insertion of a 1.1 kb gene expression cassette into targeted genomic locations.
• CREATE system operates without inducing double-strand breaks or requiring DNA templates for gene integration.
• Mechanistic studies confirmed high editing specificity with no detectable off-target events.

Conclusions:

• CREATE represents a programmable, RNA-based gene delivery technology with significant therapeutic potential for genetic diseases.
• The system overcomes limitations of traditional genome editing by enabling template-free, precise insertion of large genes.