An Antibody‐CRISPR/Cas Conjugate Platform for Target‐Specific Delivery and Gene Editing in Cancer

Abstract The CRISPR/Cas system has been introduced as an innovative tool for therapy, however achieving specific delivery to the target has been a major challenge. Here, an antibody‐CRISPR/Cas conjugate platform that enables specific delivery and target gene editing in HER2‐positive cancer is introduced. The CRISPR/Cas system by replacing specific residues of Cas9 with an unnatural amino acid is engineered, that can be complexed with a nanocarrier and bioorthogonally functionalized with a monoclonal antibody targeting HER2. The resultant antibody‐conjugated CRISPR/Cas nanocomplexes can be specifically delivered and induce gene editing in HER2‐positive cancer cells in vitro. It is demonstrated that the in vivo delivery of the antibody‐CRISPR/Cas nanocomplexes can effectively disrupt the plk1 gene in HER2‐positive ovarian cancer, resulting in substantial suppression of tumor growth. The current study presents a useful therapeutic platform for antibody‐mediated delivery of CRISPR/Cas for the treatment of various cancers and genetic diseases.

Analysis by SDS-PAGE and coomassie blue staining (left) and fluorescence detection (right).

Figure S2 .
Figure S2.Purification of Cas9aha.SDS-PAGE analysis during a) affinity chromatography and b) size exclusion chromatography.

Figure S3 .
Figure S3.Bioorthogonal reactivity of bacterial lysates after IPTG induction.Lysates from B834(DE3) were reacted with DBCO-BDP-FL and SDS-PAGE was performed for analysis by coomassie blue staining (left) and fluorescence detection (right).Bacterial lysates from growth in Met before IPTG induction (Lane 1); growth in Met after IPTG induction (Lane 2); growth in AHA before IPTG induction (Lane 3); growth in AHA after IPTG induction (Lane 4).

Figure S7 .
Figure S7.Characterization of Cas9aha RNPs reacted with DBCO-αHer by SDS-PAGE.a) Various incubation times used for reaction, b) Conjugation efficiency depending on the molar ratio of Cas9aha RNP:DBCO-⍺Her, and c) comparison with native Cas9 RNPs reacted with Mal-αHer (Cas9 RNP+Mal-αHer).Conjugation efficiencies were calculated by image analysis of band intensities and labeled below.

Figure S8 .
Figure S8.Analysis of ⍺Her-Cas9aha RNPs incubated in reducing condition (5 mM GSH) by SDS-PAGE and Coomassie blue staining.

Figure S16 .
Figure S16.Examination of co-localization of Cas9aha and Herceptin in SKOV3 cells treated with ⍺Her-CrNC.a) Confocal images of the treated cells (red: Cas9aha; cyan: Herceptin; blue: DAPI; green: actin, 40X magnification, scale bar: 50 µm), and b) relative fluorescence intensity scan along the arrow line indicated in a).

Figure S19 .
Figure S19.a) Biodistribution of ⍺Her-CrNC and controls after intravenous injection, by detecting AF750 the fluorescence of Cas9aha (24 h post-injection), and b) quantification of average radiant efficiency from a).

Figure S20 .
Figure S20.Apoptosis levels of tumors after in vivo delivery of complexes.αHer-CrNC (plk1) and the control complexes were treated to SKOV3 tumors in mice, harvested on day 19, and analyzed by the Annexin V/PI apoptosis assay.

Table S1 .
Sequences of oligonucleotides for target DNA, sgRNAs, and primers.

Table S3 .
Components of culture media for bacterial expression of Cas9aha.