Azide-functionalized SpCas9 enables generation of site-selective and bioactive Cas9-siRNA conjugates

Abstract

The gene editing enzyme SpCas9 was engineered to display azide on its surface, enabling azide–alkyne click conjugation. As a proof of concept, siRNA functionalized with a reduction sensitive linker and ring-strained alkyne tetramethylthiocycloheptyne sulfoximine was conjugated to SpCas9 on four different residues with varying efficiency and retained protein activity. Conjugation to residue 539 was successful and site-selective while retaining SpCas9 and siRNA bioactivity.