Modular click-chemistry-based engineering minimally perturbs CRISPR ribonucleoprotein formation for genome imaging applications

CRISPR-Cas9 serves as a crucial tool for genome imaging due to its ability to bind DNA specifically. Researchers have devised complex modifications to CRISPR-Cas9, often relying on expression-based delivery. However, these strategies frequently disrupt the structure, and their effectiveness is contingent upon vector design and in vivo CRISPR ribonucleoprotein (RNP) assembly. Previous research has primarily concentrated on refining the expression-based delivery systems, leaving the core engineering process underexplored.

We have devised a modular SPAAC (bio-orthogonal strain-promoted azide-alkyne cycloaddition) click-chemistry-based approach to engineer CRISPR RNP, which operates independently of assembly. This strategy allows for the modification of CRISPR with fluorophores and DNA at various stages of RNP in vitro assembly. We have tested this technology for genome imaging purposes.

Our findings indicate that engineering the native CRISPR RNP after its formation reduces structural perturbations and results in a twofold increase in the accessibility of the clicked DNA.

Furthermore, we have employed SPAAC to attach non-native RNA-DNA hybrids, irrespective of their 5' to 3' directionality, thereby creating a covalently-attached rolling-circle signal amplification approach. The modularity and versatility of our method hold the promise of significant advancements in both CRISPR imaging and prime-editing domains.