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

Abstract

The gene editing enzyme Cas9 derived from S.pyogenes (SpCas9) was engineered for regioselective azide-alkyne click conjugation of synergistic therapeutic molecules. As a proof of concept, siRNA functionalized with a reduction sensitive linker and the ring-strained alkyne tetramethylthiocycloheptyne sulfoximine was conjugated to SpCas9 on four different amino acids with varying efficiency and retained protein activity. Conjugation at amino acid 539 was succesful, site-selective, and both SpCas9 and the conjugated siRNA showed bioactivity in vitro.