Although CRISPR-Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate-DNA oligos (PSs), that one can efficiently deliver single and bi-specific CRISPR-Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2+ ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.

Figure S1. Increased TLR9 expression in induced glioma spheres. Figure S2. TLR9 and STAT3 form a feed-forward loop in GSCs. Figure S3. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits tumor growth. Figure S4. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits GSCs. Figure S5. Targeting Brain tumors systemically with CpG-siRNA reaches the tumor site.

Supplementary Figure 6 from Toll-like Receptor 9 Activation of Signal Transducer and Activator of Transcription 3 Constrains Its Agonist-Based Immunotherapy

Suppl. Fig. S3 showing CTLA4 expression by tumor associated CD19 B cells

Suppl. Fig. S1 showing CTLA4 expression and CD86 cellular internalization by human BCL and human MM cells acquired by flow cytometry.

Supplementary Figure 1 from Toll-like Receptor 9 Activation of Signal Transducer and Activator of Transcription 3 Constrains Its Agonist-Based Immunotherapy

<p>Suppl. Fig. S2 showing improved Th1 and Th2 maturation and increased CD3 T cell activation upon CTLA4 blockade in vivo.</p>

Supplementary Data from Antitumor Activity of Targeting Src Kinases in Endothelial and Myeloid Cell Compartments of the Tumor Microenvironment

Supplementary Figure 1 from Sunitinib Inhibition of Stat3 Induces Renal Cell Carcinoma Tumor Cell Apoptosis and Reduces Immunosuppressive Cells

Supplementary Figure 2 from Toll-like Receptor 9 Activation of Signal Transducer and Activator of Transcription 3 Constrains Its Agonist-Based Immunotherapy

PDF file - 5546K, Supplementary Fig.2: STAT3 activation is associated with upregulated CSC marker expression.

Supplementary Figure 2 from Targeting Stat3 in the Myeloid Compartment Drastically Improves the In vivo Antitumor Functions of Adoptively Transferred T Cells

PDF file - 1025K, Supplementary Fig. 1: Elevated STAT3 activity results in enhanced tumor growth.

Supplementary Figure 2 from Targeting STAT3 in Adoptively Transferred T Cells Promotes Their In Vivo Expansion and Antitumor Effects

PDF file - 2401K, Supplementary Fig. 3: Characterization of recombinant IL-6RFP.

Supplementary Figure 1 from Targeting Stat3 in the Myeloid Compartment Drastically Improves the In vivo Antitumor Functions of Adoptively Transferred T Cells

Supplementary Figure 7 from Toll-like Receptor 9 Activation of Signal Transducer and Activator of Transcription 3 Constrains Its Agonist-Based Immunotherapy

Supplementary Figure 4 from Toll-like Receptor 9 Activation of Signal Transducer and Activator of Transcription 3 Constrains Its Agonist-Based Immunotherapy

Supplementary Figure 5 from Toll-like Receptor 9 Activation of Signal Transducer and Activator of Transcription 3 Constrains Its Agonist-Based Immunotherapy