Coiled-coil mediated tethering of CRISPR/CAS and exonucleases for enhanced genome editing
The global gene editing market is propelled by various applications, ranging from the discovery of new pharmaceuticals to genetic engineering of plants and microorganisms. Also popular are accessible gene editing tools. CRISPR/Cas is an important one due to its multiplexing ability and relatively easy design. Our technology improves gene editing with CRISPR/Cas by enabling the modification of a larger part of the genome.
Our technology builds on CRISPR/Cas with tethering – the attachment of a molecule to the Cas9 protein. In our case, the molecule tethered to Cas9 was exonuclease III (EXOIII). We achieved tethering in a cell using heterodimeric peptide pairs linked to Cas9 and EXOIII, which form a coiled-coil due to hydrophobic and electrostatic interactions between the peptides. Cas9 subsequently brings EXOIII into close proximity of the double stranded breaks (DSB). This allows for additional DNA recession, leading to different 5' overhangs with greater mismatches in template and non-template DNA strands, resulting in higher indel mutations.
Our technology allows for a simple tethering of various exonucleases with Cas9 and its variants via coiled-coils. This improves DNA recession and enables a higher percentage or degree of genome modification. No additional increase in off-target effects has been observed and the technology does not induce cytotoxicity.
Developed by: Department of Synthetic Biology and Immunology.
Technology readiness level: TRL3.
Status of intellectual property:Patent granted.
Cooperation opportunities: Joint development, intellectual property licensing, or sale.
2020 NICKI Support Recipient
Scientific and academic papers
Lainšček, D., Forstnerič, V., Mikolič, V. et al. Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing. Nat Commun 13, 3604 (2022). https://doi.org/10.1038/s41467-022-31386-1.