Cysteine‐independent CRISPR‐associated protein labeling for presentation and co‐delivery of molecules toward genetic and epigenetic regulations

Labeling of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins (Cas) remains an immense challenge for their genome engineering applications. To date, cysteine‐mediated bioconjugation is the most efficient strategy for labeling Cas proteins. However, introducing a cysteine residue in the protein at the right place might be challenging without perturbing the enzymatic activity. We report a method that does not require cysteine residues for small molecule presentation on the CRISPR‐associated protein Sp Cas9 for in vitro protein detection, probing cellular protein expression, and nuclear co‐delivery of molecules in mammalian cells. We repurposed a simple protein purification tag His6 peptide for non‐covalent labeling of molecules on the CRISPR enzyme Sp Cas9. The small molecule labeling enabled us to rapidly detect Sp Cas9 in a biochemical assay. We demonstrate that small molecule labeling can be utilized for probing bacterial protein expression in realtime. Furthermore, we coupled Sp Cas9’s nuclear‐targeting ability in co‐delivering the presenting small molecules to the mammalian cell nucleus for prospective genome engineering applications. Furthermore, we demonstrate that the method can be generalized to label oligonucleotides for multiplexing CRISPR‐based genome editing and template‐mediated DNA repair applications. This work paves the way for genomic loci‐specific bioactive small molecule and oligonucleotide co‐delivery toward genetic and epigenetic regulations.