Effective Genome Editing Using CRISPR‐Cas9 Nanoflowers

Abstract

CRISPR-Cas9 as a powerful gene-editing tool has tremendous potential for the treatment of genetic diseases. Herein, a new mesoporous nanoflower (NF)-like delivery nanoplatform termed Cas9-NF is reported by crosslinking Cas9 and polymeric micelles that enables efficient intracellular delivery and controlled release of Cas9 in response to reductive microenvironment in tumor cells. The flower morphology is flexibly tunable by the protein concentration and different types of crosslinkers. Cas9 protein, embedded between polymeric micelles and protected by Cas9-NF, remains stable even under extreme pH conditions. Responsive cleavage of crosslinkers in tumor cells, leads to the traceless release of Cas9 for efficient gene knockout in nucleus. This crosslinked nanoparticle exhibits excellent capability of downregulating oncogene expression and inhibiting tumor growth in a murine tumor model. Taken together, these findings pave a new pathway toward the application of the protein-micelle crosslinked nanoflower for protein delivery, which warrants further investigations for gene regulation and cancer treatment.

CRISPR/Cas9 and Chlorophyll Coordination Micelles for Cancer Treatment by Genome Editing and Photodynamic Therapy

“…Previously we reported that A549 cells could also be killed by cutting the genes of polo‐like kinase 1 (PLK‐1) and we used this paradigm as a control of single gene therapy using Cas9‐PLK1‐Chl@F127. [ 45,46 ] As shown in Figure 4d, the synergistic therapeutic treatment time was decreased by 48 h compared with the sole gene editing therapy. The therapeutic killing effect by Cas9‐APE1‐NRF2‐Chl@F127 for 24 h was similar to that of Cas9‐PLK1‐Chl@F127 micelles treated for 72 h, indicative of the advantage of shortened treatment time.…”

Design of Liposome Formulations for CRISPR/Cas9 Enzyme Immobilization: Evaluation of 5-Alpha-Reductase Enzyme Knockout for Androgenic Disorders

“…59 Direct delivery of Cas9 RNPs has advantages over plasmid or mRNA delivery methods, including reduced off-target effects, low toxicity, and high editing efficiency. 60,69 To ensure effective genome editing, it is critical for Cas9 RNPs to be timely released in the cytosol and enter the nucleus after cell internalization. 60 In this context, the EE and LC of the optimal formulation were investigated.…”

“…60,69 To ensure effective genome editing, it is critical for Cas9 RNPs to be timely released in the cytosol and enter the nucleus after cell internalization. 60 In this context, the EE and LC of the optimal formulation were investigated. Accordingly, the EE was determined to be 80.6% and the LC was determined to be 12.59%.…”

“…Studies have shown that the release of Cas9 RNPs is more favorable under acidic conditions, similar to the endosomal environment, with a higher percentage released at pH 5 compared to those at pH 6 and pH 7.4 . Direct delivery of Cas9 RNPs has advantages over plasmid or mRNA delivery methods, including reduced off-target effects, low toxicity, and high editing efficiency. , To ensure effective genome editing, it is critical for Cas9 RNPs to be timely released in the cytosol and enter the nucleus after cell internalization . In this context, the EE and LC of the optimal formulation were investigated.…”

Biomaterial-assisted targeted and controlled delivery of CRISPR/Cas9 for precise gene editing

“…The system excellently downregulated the oncogene and suppressed the tumor growth. 22 2-(Diisopropylamino) ethyl methacrylate and folic acid (FA) modified trimethyl chitosan (TMC) NPs for survivin shRNA-expressing plasmid or survivin CRISPR/Cas9 plasmid, and doxorubicin (DOX) increased the uptake of pDNA and DOX into cancer cells and triggered the release under an acidic pH response 23 and provided a promising modality for cancer treatment. PEGylated polyplex micelles co-encapsulating Cas9 mRNA and sgRNA showed high genome editing efficiency owing to the increased diffusion of PEGylated polyplex micelles in the brain.…”