Orthogonal Chemical Activation of Enzyme-Inducible CRISPR/Cas9 for Cell-Selective Genome Editing

Abstract

The precision and therapeutic potential of CRISPR/Cas9 genome editing are greatly challenged by the less control over Cas9-mediated DNA cleavage. Herein, we introduce a conditional and cell-selective genome editing system controlled by disease-associated enzymes, termed enzyme-inducible CRISPR (eiCRISPR). eiCRISPR comprises Cas9 protein, a self-blocked inactive single-guide RNA (bsgRNA), and a chemically caged deoxyribozyme (DNAzyme) that activates bsgRNA and eiCRISPR in a controllable manner. We design chemical modifications of DNAzyme to suppress its ability to cleave the blocking region of bsgRNA, while the decaging of DNAzyme triggered by the tumor cell-overexpressed enzyme, for instance, NAD(P)H:quinone oxidoreductase (NQO1), restores the activity of bsgRNA and switches on eiCRISPR selectively for genome editing in cancer cells. Moreover, using a biodegradable lipid nanoparticle to deliver eiCRISPR in a tumor-bearing xenograft, we show that the in vivo activation of eiCRISPR enables the editing of human papillomavirus 18 E6 for potential cancer therapy. The strategy of postsynthetic and site-specific modification of DNAzyme is compatible with endogenous chemistries for regulating eiCRISPR for cell-selective genome editing and targeted gene therapy.

High‐Precision Synthesis of RNA‐Loaded Lipid Nanoparticles for Biomedical Applications

“…Moreover, Cai et al successfully demonstrated a spatiotemporally controllable cell-selective genome editing technique using biodegradable lipid-based LNP (Figure 13f). [317] The genome editing system is regulated by enzyme-inducible CRISPR/Cas9 (eiCRISPR) comprising of Cas9 protein, self-blocked inactive single-guide RNA (bsgRNA), and deoxyribozyme (DNAzyme). DNAzyme could be activated by upregulated NAD(P)H:quinone oxidoreductase (NQO1) signals in the disease cells, which cleaves the blocking region of bsgRNA, and further activates eiCRISPR for tumor cell-selective genome editing.…”

Intracellular Delivery of mRNA for Cell‐Selective CRISPR/Cas9 Genome Editing using Lipid Nanoparticles

“…This strategy reduces the negative effects of genome editing technology and improves the therapeutic potential of genome editing technology (Figure 6). [11] …”

“…[6] Therefore, mRNA holds great promise for developing biotherapeutics for treating a large variety of genetic disorders, including protein replacement therapy, [7] mRNA vaccines, [8,9] immunotherapies, [10] and gene editing. [11] mRNA is a large, negatively charged biomacromolecule that faces significant challenges in attempting to penetrate cell membranes spontaneously. In addition, mRNA is prone to rapid degradation in various biological settings, such as blood and other body fluids.…”

An orthogonally activatable CRISPR-Cas13d nanoprodrug to reverse chemoresistance for enhanced chemo-photodynamic therapy

“…6 For example, clustered regularly interspaced short palindromic repeatsassociated protein 9 (CRISPR-Cas9) technology has been found to reverse MDR mediated by ATP-binding cassette (ABC) transporters with a significantly higher outcome than other gene editing technologies due to its simple design, flexible target region, higher editing efficiency and multiplexing. [7][8][9][10] Despite great advances, the majority of CRISPR-Cas9 systems still suffer from some formidable problems, including off-target genomic alterations and genotoxicity, potential immunological risk by Cas9-specific T-cells, and unsatisfactory targeted delivery. 8 To address this challenge, CRISPR-Cas13d, a RNAguided Type VI Cas protein, has been identified for target gene knockdown without altering the genome.…”