Enhanced RNA-templated prime editing

This technology is an RNA templated genome editing technique that can be utilized in any organism, without the typical requirement for homologous recombination.

Unmet Need: Genome editing without homologous recombination

Current CRISPR-based methods to edit the genome are dependent on homology-directed repair (HDR) following double-strand DNA breaks, which may result in non-target deletions and insertions, translocations, and rearrangements. Additionally, they are less efficient in non-dividing cells. Although some techniques have been described that circumvent these limitations, available CRISPR gene editing methods could be improved in terms of efficiency and accuracy.

The Technology: RNA-templated prime editing with enhanced RT recruitment

This technology is a strategy for creating single-strand breaks in DNA to introduce point mutations for faster, more accurate genomic modifications. It uses a Cas9-nickase-based prime editor that nicks one DNA strand and copies an edit from an RNA template embedded in an engineered prime-editing guide RNA (pegRNA), enabling precise substitutions, insertions, and deletions without double-strand breaks. The system combines several components, including a cas9 nickase (nCas9), a reverse transcriptase fused to Cas9, and an extended guide RNA containing an RNA template for reverse transcription that includes the desired mutations, plus a primer-binding site that initiates cDNA synthesis. This method eliminates the need for double-stranded DNA breaks, is more efficient at successfully introducing mutations, and can be used for non-dividing cells, further expanding the applications and addressing the shortcomings of the ubiquitous CRISPR-Cas9 technology.

This technology has been validated with human cancer cell lines and primary non-dividing neurons.

Applications:

• Modification of cells for research
• Genetic engineering for cell therapies
• Modification of organisms for research, agriculture, and husbandry
• In vivo or ex vivo therapeutic correction of pathogenic variants in hard-to-edit cells (e.g., neurons, hepatocytes)

Advantages:

• Higher efficiency
• Improved accuracy with minimal indel formation
• Enables gene editing of non-dividing cells
• Allows for modification of longer sequences
• Modular RT recruitment/fusion allows larger edits and rapid customization across organisms

Lead Inventor:

Alejandro Chavez, M.D., Ph.D.

Patent Information:

Patent Pending (US20220411768)

Related Publications:

Tech Ventures Reference:

• IR CU20126

• Licensing Contact: Cynthia Lang