Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Genome editing technologies offer powerful tools for genetic modification.
• DNA-dependent polymerases (DDPs) present unique advantages for installing diverse edits.
• Existing methods may have limitations in precision or scope of edits.

Purpose of the Study:

• To develop a novel genome writing platform for precise and versatile genome editing.
• To couple the benefits of DDPs with RNA-programmable nickases for enhanced editing capabilities.
• To enable the installation of a range of edits, including substitutions, insertions, and deletions.

Main Methods:

• Development of the 'click editing' (CE) platform.
• Utilizing HUH endonucleases for 'click'-like bioconjugation of DNA templates.
• Employing RNA-programmable nickases to target specific genomic loci.
• Iterative optimization of CE components and 'click DNA' (clkDNA) templates.
• Screening of clkDNA modifications to enhance editing efficiency.

Main Results:

• Demonstrated precise installation of substitutions, insertions, and deletions at targeted genomic loci.
• Achieved precise editing efficiencies of up to approximately 30% in diverse human cell types.
• Minimized unwanted insertions and deletions (indels) at editing sites.
• Showcased improved editing efficiency through screening of modified clkDNA oligonucleotides.
• Validated the versatility of click editing in immortalized human cell lines and primary fibroblasts.

Conclusions:

• Click editing is a precise and versatile genome editing platform.
• The technology leverages DDPs and RNA-programmable nickases for diverse genomic modifications.
• Click editing offers a promising approach for various biological applications requiring accurate genome alterations.