Search

Amplifying intracellular oxidative stress by organelle‐targeted reactive oxygen species (ROS) production combined with tumor cell‐specific gene disruption is a promising strategy for tumor treatment. However, due to the vulnerability of CRISPR/Cas9 ribonucleoproteins (RNPs) to ROS, co‐delivery of CRISPR/Cas9 RNPs and ROS generators to enhance the sensitivity of tumor cells to oxidative stress remains challenging. Herein, a cascade‐responsive "oxidative stress amplifier" (named DR‐TAF‐pHT/FA) is proposed, which can successively respond to cathepsin B, localized laser irradiation and ATP to generate ROS on the lysosomal membrane of tumor cells and release Cas9/sgNrf2 RNPs for efficient gene disruption. It is demonstrated that, under near infrared (NIR) irradiation, DR‐TAF‐pHT/FA achieves targeted rupture of lysosomal membranes, inducing significant intracellular oxidative stress. Meanwhile, due to the protective function of TAF coating (TA‐Fe3+ coordination self‐assembled networks), Cas9/sgNrf2 RNPs can safely escape into the cytoplasm and be released in response to ATP, further amplifying oxidative stress and promoting tumor cell apoptosis through efficient Nrf2 gene disruption. Treatment with DR‐TAF‐pHT/FA + NIR significantly improves tumor ablation efficiency and extends median survival time (over 70 days) in Hela xenograft models. This "oxidative stress amplifier" provides a new paradigm for multimodal and synergistic tumor therapy through precise lysosomal membrane bursting together with efficient Nrf2 gene disruption. [ABSTRACT FROM AUTHOR]

Deoxyribonucleic acid (DNA) methylation is one of the epigenetic characteristics that result in heritable and revisable phenotype changes but without sequence changes in DNA. Aberrant methylation occurring at a specific locus was reported to be associated with cancers, insulin resistance, obesity, Alzheimer's disease, Parkinson's disease, etc. Therefore, locus‐specific DNA methylation can serve as a valuable biomarker for disease diagnosis and therapy. Recently, Clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas systems are applied to develop biosensors for DNA, ribonucleic acid, proteins, and small molecules detection. Because of their highly specific binding ability and signal amplification capacity, CRISPR‐Cas assisted biosensor also serve as a potential tool for locus‐specific detection of DNA methylation. In this perspective, based on the detection principle, a detailed classification and comprehensive discussion of recent works about the latest advances in locus‐specific detection of DNA methylation using CRISPR‐Cas systems are provided. Furthermore, current challenges and future perspectives of CRISPR‐based locus‐specific detection of DNA methylation are outlined. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeats (CRISPR) system provides a new molecular diagnostic tool for construction of biosensor platforms due to its high programmability and target specificity. Herein, we developed a CRISPR-empowered electrochemical biosensor by combining the advantages of CRISPR/Cas13a and primer exchange reaction (PER), named PER-E-CRISPR, for target amplification-free and sensitive detection of miR-21. Dual-signal amplification procedures involve the binding of target miR-21 induced by CRISPR-based amplification, along with the hybridization of multiple short single-stranded DNA strands with PER concatemers. When target miR-21 is present, CRISPR/Cas13a specifically recognizes the target miRNA, triggering the trans -cleavage activity of CRISPR/Cas13a. Then Cas13a/crRNA/miRNA cleaved the predesigned ribonucleotide site in hairpin 1 (HP1) and released trigger to open hairpin 2 (HP2) modified on the electrode surface. Then PER bridge sequence contained in HP2 is exposed and hybridized with PER concatemers, following multiple short single-stranded DNA tagged with methylene blue (ssDNA-MB) bond with the PER concatemers. Under optimized conditions, PER-E-CRISPR assay for detecting miR-21 exhibits linearity in dynamic range from 10−13 to 10−7 M, and we obtained a limit of detection (LOD) of 30.2 fM. The established PER-E-CRISPR biosensor shows perfect practical performance in actual plasma, which may have great promising prospects for miRNA detection in the field of molecular diagnosis. [Display omitted] • CRISPR-empowered electrochemical biosensor with dual-signal amplification. • Nucleic acid amplification-free and sensitive detection of miRNA by PER-E-CRISPR. • Pre-synthesis of PER concatemer significantly improve the detection signal. [ABSTRACT FROM AUTHOR]

Nanoparticle-based CRISPR/Cas9 delivery systems hold great promise for specific and precise treatment of genetic disorder diseases. Herein, we developed a DNA nanoflower-based platform for microRNA-responsive cytosolic delivery of Cas9/sgRNA complex into tumor cells. The biocompatible DNA nano-vehicles can efficiently load Cas9/sgRNA by sequence hybridization. Importantly, this hybridization can be replaced by a tumor specific miRNA through toehold-mediated strand displacement process and achieve cell-type-specific release of Cas9/sgRNA from the DNA nanoflowers. We have verified that this miRNA-responsive releasing process can significantly improve the genome editing efficiency comparing with non-responsive control. This strategy suggests a versatile way for designing more specific and efficient CRISPR-based genome therapy system by incorporating stimuli-responsive Cas9/sgRNA release process. [ABSTRACT FROM AUTHOR]