Efficient Genome Editing Tool Using CODE Systems for Precise Modifications

Complements Existing Prime Editors to Expand Toolbox for Genome Manipulations

This precision-driven genome editing tool uses chimeric oligonucleotide-directed editing (CODE) to replace, insert, or delete specific genes with exceptional accuracy. The genome editing market, valued at nearly $10 billion in 2024 and anticipated to grow to $25 billion by 2030, faces significant hurdles. Traditional prime editing, while powerful, faces challenges such as varied efficiency across genomic loci, competition between edited and unedited DNA flaps, and inaccuracies like reverse transcriptase overextension.

University of Florida researchers have engineered nCas9 DNA polymerase fusion proteins for CODE systems, which improve efficiency and precision of genome editing by integrating a DNA-dependent DNA polymerase with a chimeric pegRNA. The approach addresses key limitations with traditional prime editing. This genome editing tool is promising for therapeutic applications, offering versatility for precise genome modifications without the need for double-stranded breaks.

Application

Genome editing tool for precise, efficient genome manipulation for gene therapy, research tools, drug discovery and development, and more

Advantages

• Leverages DNA-dependent DNA polymerases, offering high processivity, proofreading capabilities, and minimal reverse transcriptase activities, enhancing editing efficiency and reducing off-target effects
• Uses a nickase Cas9-DNA polymerase fusion protein and a chimeric pegRNA, which contains both RNA and DNA bases, facilitating precise genome modifications without double-stranded breaks
• Incorporates a robust Bst DNA polymerase derivative, achieving high editing efficiency with low unintended edits
• Can be delivered via plasmids or RNP complexes, offering versatility for therapeutic applications
• Provides for cost-effective ligation-based methods for synthesizing chimeric pegRNAs, enhancing practicality and scalability

Technology

This cutting-edge genome editing tool consists of unique chimeric guides and nickase enzymes that precisely target and replace desired sections of DNA. The system's robust capabilities, stemming from the combination of DNA polymerases and engineered proteins, reduce auto-inhibition and leverage the unique properties of DNA polymerases, such as high processivity and proofreading capabilities, to enhance editing efficiency and accuracy. The development of CODEMax and CODEMax(exo+) systems, engineered with a robust Bst DNA polymerase derivative, further improves gene correction efficiency. These systems perform small insertions, deletions, and substitutions more effectively than traditional prime editors, expanding the toolbox for genome manipulation without double-stranded breaks.