CMN Weekly (12 July 2024) - Your Weekly CRISPR Medicine News

Top picks

• A team of American researchers have used CRISPR-Cas to target and correct the MIR96 mutation 14C>A in adult mice with DFNA50, a form of delayed-onset hearing loss. The KKH variant of Staphylococcus aureus Cas9 (SaCas9-KKH) and optimised sgRNA were delivered via AAV to the cochleae of mutant mice, resulting in significant long-term hearing improvement in both presymptomatic and symptomatic mice. The dual-AAV system using sgRNAs targeting all human MIR96 mutations demonstrated efficient, specific editing, highlighting a potential treatment for MIR96-related deafness.
• Paris-based Eligo Bioscience has achieved a significant breakthrough in microbiome editing, engineering a phage-derived particle to deliver a base editor for modifying Escherichia coli in the mouse gut. Editing a β-lactamase gene achieved 93% efficiency in the target bacterial population with a single dose. Edited bacteria were stable for at least 42 days. This non-replicative DNA vector approach also edited therapeutic genes in E. coli and Klebsiella pneumoniae in vitro, demonstrating the potential for microbiome-targeted therapies. See also our summary of the breakthrough with comments from the CEO of Eligo Bioscience, Xavier Duportet.

Research

• British researchers have used CRISPR-Cas9 for saturation genome editing (SGE) on BAP1, a gene linked to tumour development and neurodevelopmental issues, identifying 6,196 out of 18,108 variants with abnormal functions. They transfected HAP1 cells expressing Cas9 with sgRNA plasmids targeting specific regions of BAP1 and subsequently edited these cells with variant libraries through homologous recombination at the Cas9 cut site. Evaluating the variants in the UK Biobank and other databases found that disruptive BAP1 variants correlate with elevated IGF-1 levels, hinting at a potential pathological mechanism.
• Researchers in Australia have employed CRISPR interference (CRISPRi) to reduce nidogen-2 (NID2) in cancer-associated fibroblasts (CAFs) of pancreatic ductal adenocarcinoma (PDAC) models. This reduction led to decreased tissue stiffness, impaired cancer cell invasion, and improved vascular function. CRISPRi-modified tumours showed enhanced response to chemotherapy, reduced liver metastasis, and increased survival, identifying NID2 as a potential target for PDAC treatment.
• A new "multitron" technology uses bacterial retron arrays to precisely edit multiple genome sites simultaneously. The multitron system generates multiple donor-encoding DNAs from a single transcript, streamlining complex genomic edits. This approach is effective in both prokaryotic recombineering and eukaryotic CRISPR-Cas editing, with applications in molecular recording, genetic element minimisation, and metabolic engineering.
• A new technology, DECOR (deaminase-enabled recoding of RNA), uses an evolved E. coli TadA8e enzyme for precise adenosine-to-inosine editing at CRISPR-specified sites in the human transcriptome. DECOR shows high on-target activity with 88% fewer off-target effects compared to more conventional ADAR platforms. It successfully corrected IRF6 gene expression in Van der Woude syndrome, increasing functional transcript levels from 12.3% to 36.5%.
• David Liu and colleagues have systematically optimised prime editing (PE) to correct the CFTR F508del mutation, a leading cause of cystic fibrosis. By integrating six enhancements, they increased correction efficiency from under 0.5% in HEK293T cells to 58% in bronchial epithelial cells and 25% in patient-derived cells. This method achieved minimal off-target effects and superior edit-to-indel ratios. It restored CFTR function to over 50% of wild-type levels, suggesting potential for a durable one-time CF treatment.
• A new platform, DEBCT, offers a scalable cell therapy for Dystrophic Epidermolysis Bullosa (DEB) using CRISPR-corrected, patient-specific iPS cell-derived skin grafts. This method combines genetic correction and reprogramming, producing diverse skin cell lineages. Mouse models show disease-modifying effects and a favourable safety profile. DEBCT overcomes manufacturing and safety challenges, providing a reproducible and safe treatment for DEB.

CRISPR screens

• A genome-wide CRISPR screen in PANC-1 cells treated with nab-paclitaxel has identified spindle assembly checkpoint (SAC) genes - BUB1B, BUB3, and TTK - that enhance survival under treatment. Knockdown of these genes reduces paclitaxel-induced cell death, and inhibitors targeting TTK (BAY 1217389, MPI 0479605) have similar effects. Overexpression of these genes does not alter paclitaxel sensitivity. These findings elucidate paclitaxel cytotoxicity mechanisms, aiding in understanding and potentially improving pancreatic cancer therapies.
• A CRISPR screen in head and neck squamous cell carcinoma (HNSCC) cells revealed that PD-L1 deficiency makes cells dependent on ferroptosis-related genes. Inducing ferroptosis in PD-L1 knockout cells accelerates cell death. PD-L1 activates SOD2 to maintain redox balance, reducing ROS and ferroptosis. Targeting PD-L1's role in ferroptosis resistance could enhance cancer therapy.

Detection

• A novel rapid, visual molecular detection method for tick-borne encephalitis virus (TBEV) uses RT-recombinase-aided amplification, CRISPR/Cas13a, and lateral flow dipsticks. This method, with 50 CFU/ml sensitivity and no cross-reactivity, delivers results within an hour at 37-42°C, requiring no complex instruments. Tested on clinical samples, it achieved 100% sensitivity and specificity, matching RT-qPCR results, and shows promise as a point-of-care diagnostic for TBEV.
• New CRISPR-based SHERLOCK assays can detect RNA-fusion transcripts in fusion-driven leukemias, such as APL, Ph+ ALL, and CML. Validated on patient samples and dried blood spots, these assays showed 100% sensitivity and specificity. Optimisations enable use outside advanced labs, making rapid, point-of-care diagnostics accessible in low-resource settings. This technology can improve outcomes by facilitating timely diagnosis and treatment for these treatable cancers.
• Researchers have developed a sensitive electrochemical biosensor to detect folate receptor-positive circulating tumour cells (CTCs) in breast cancer. Folate-functionalised beads capture CTCs, and CRISPR-Cas12a cleaves DNA modified with methylene blue, amplifying the electrochemical signal. The biosensor detects as few as two cells/mL with high sensitivity and stability, offering a promising tool for CTC analysis.
• A new method for detecting lncRNA RMRP in urine exosomes from bladder cancer (BCa) patients uses RT-RAA and CRISPR-Cas12a. This technique enhances diagnostic specificity, reduces testing time to 30 minutes, and offers real-time detection via blue light. It surpasses traditional RT-qPCR in accuracy and sensitivity. The RT-RAA-CRISPR/Cas12a method is rapid, sensitive, user-friendly, and promising for clinical BCa diagnosis.
• A novel fluorescein-tetramethylrhodamine (FAM-TAMRA) FRET-based substrate for CRISPR-Cas12a biosensors enhances signalling and reduces detection time without altering assay setups. This dual-signal substrate outperformed traditional FAM-BHQ reporters and was successfully used in an RPA-CRISPR-Cas12a DETECTR assay to detect Human papillomavirus in patient samples, demonstrating its superior utility.

Reviews

• Recent Advances in RNA Interference-Based Therapy for Hepatocellular Carcinoma: Emphasis on siRNA. This review discusses the advancements and challenges of siRNA therapies for hepatocellular carcinoma. It focuses on their roles in inhibiting angiogenesis, reducing cell proliferation, promoting apoptosis, and addressing delivery and immunogenic issues.