Authors
Van Tricht, C., Thonon, S., Wouters, D., Sifrim, A., Vandereyken, K., Pinheiro, V. B., Voet, T., Lammertyn, J., Spasic, D.
Abstract
CRISPR-Cas9 has become a key genome imaging tool, because of its sequence-specific binding to genomic DNA. To achieve signal amplification or multiplexing, various complex CRISPR-Cas9 modifications were introduced, primarily relying on expression-based delivery. However, these engineering strategies often invoke structural perturbations and their performance remained dictated by vector design and in vivo CRISPR ribonucleoprotein (RNP) assembly. To date, research mainly focused on optimizing the expression-based delivery mechanisms of CRISPR components rather than addressing the engineering itself. Here, we developed a modular SPAAC (bio-orthogonal strain-promoted azide-alkyne cycloaddition) click-chemistry-based strategy for engineering the CRISPR RNP that is completely dissociated from its assembly. We demonstrated that CRISPR can be modified (with fluorophores and DNA) at all stages of RNP in vitro assembly and subsequently validated the technology for genome imaging. Importantly, we also revealed that engineering native CRISPR RNP only after it is being formed, minimizes structural perturbations to the complex and yields a two-fold enhanced accessibility of 'clicked' DNA. Finally, we utilized SPAAC to conjugate non-native RNA-DNA hybrids, regardless of 5'-3' directionality, to develop a covalently-attached rolling-circle signal amplification approach. The modularity and universal nature of our approach holds the potential to deliver significant advances to both CRISPR imaging and prime-editing fields.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 14 Sep 2026.
Advertisement
Stats
- Recommendations n/a n/a positive of 0 vote(s)
- Views 8
- Comments 0