Bioorthogonal CRISPR/Cas9‐Drug Conjugate: A Combinatorial Nanomedicine Platform

Abstract Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self‐delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi‐functionalized Cas9 with a drug and polymer can form self‐condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti‐cancer drug olaparib and targeting the RAD52 gene, leading to significant anti‐tumor effects in BRCA‐mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.


Table of Contents
Primer sequences used for DNA cloning of plasmids for Cas9-AzF expression.
Table S1.Plasmid information and primer sequences used for DNA cloning.

Figure S3 .Figure S4 .
Figure S3.Conjugation efficiencies of olaparib and CP reacted with Cas9-AzF.Residual azido and sulfhydryl groups were measured by a fluorescence tagging method.

Figure S7 .
Figure S7.Gene editing patterns by treatment of ComBiNE.Deletion and insertion ratios (%) in the RAD52 gene obtained by targeted deep sequencing of HCC1937 cells, after treatment with ComBiNE and Cas9-CP RNPs targeting RAD52 for 48 h at 500 nM.

Figure S18 .
Figure S18.Anti-proliferation effect upon treatment of free olaparib, Cas9-CP RNPs, and their mixture.Complexes and controls were treated to HCC1937 cells for 72 h.Cas9-CP conjugates were prepared by reacting the sulfhydryl groups of Cas9 with CP, complexed with sgRNA (1:1), and treated at 500 nM.

Figure S21 .
Figure S21.Indel frequencies of mice tumors at 24 days post-treatment.Mice with HCC1937 tumors were treated with ComBiNE and control formulations, and tissues were harvested at day 24, followed by targeted deep sequencing (n = 3).