CMN Weekly (30 August 2024) - Your Weekly CRISPR Medicine News

Some of the best links we picked up around the internet

By: Gorm Palmgren - Aug. 30, 2024

Top picks

Researchers at Cellectis have developed "Smart CAR T-cells" using TALEN gene editing to overcome challenges in solid tumour treatment. These cells feature a FAPCAR, a CAR targeting fibroblast activation protein (FAP) on cancer-associated fibroblasts (CAFs), which triggers a secondary CAR against tumour antigens like mesothelin. This dual-targeting strategy enhances anti-tumour efficacy while reducing off-target toxicity, showcasing TALEN's potential in creating safer, more effective CAR-T therapies for solid tumours.
Plofsomes (polymer-locking fusogenic liposomes) are a new vehicle for delivering RNAi or CRISPR–Cas9 complexes across the blood-brain barrier, specifically targeting glioblastoma (GBM) cells with high reactive oxygen species levels. By using these tools to suppress midkine (MDK), Plofsomes reduce temozolomide resistance and inhibit tumour growth. Crucially, their activity is confined to tumour sites, enhancing the safety of combined RNAi and CRISPR-Cas9 therapies for GBM.

Research

German researchers have developed an automated CRISPR-Cas9 genome editing process using the StemCellFactory platform, integrating a 4D-Nucleofector with a 96-well shuttle device. This system optimizes single-cell culturing and automates genome editing workflows, achieving indel rates of up to 98%. It also uses the CellCelector to monitor monoclonal colony growth, ensuring high precision. This scalable, standardized platform offers a powerful tool for large-scale studies in disease modelling, drug development, and personalized medicine.
In a not-yet peer-reviewed paper, researchers have employed CRISPR base editing to introduce revertant mutations in cis with the F508del mutation in the CFTR gene, which is the most common cause of cystic fibrosis (CF). This strategy successfully restored CFTR protein folding and function in CF cellular models, including primary epithelial cells from CF patients. The recovery of ion transport was fully validated in patient-derived epithelia, and combining this approach with small molecule therapies further enhanced F508del activity, indicating potential for combinatorial CF treatments.
South Korean researchers have revealed the first structure of the standalone Type I-C Cas3 enzyme from Neisseria lactamica (NlaCas3). Structural analysis showed that the histidine–aspartate (HD) nuclease site in NlaCas3 binds two Fe2+ ions, inhibiting its activity. However, NlaCas3 cleaves single- and double-stranded DNA in the presence of Ni2+ or Co2+, with optimal activity when both Ni2+ and Mg2+ ions are present.
A new minimal type I-F2 CRISPR-Cas system is capable of transcriptional activation and base editing in human cells and opens up new possibilities for more efficient genome manipulation in eukaryotic cells. The CRISPR-Cas system is derived from Moraxella osloensis and shows strong potential for transcriptional regulation and base editing in human cells.
Researchers have investigated the thermodynamics of CRISPR-Cas9 interactions using isothermal titration calorimetry and microscale thermophoresis. Their findings reveal that the binding of sgRNA to Cas9 is entropy-driven, compensating for unfavourable enthalpy, while the interaction between the CRISPR RNP complex and target DNA is enthalpy-driven, offsetting unfavourable entropy. Both interactions exhibit negative heat capacity changes, suggesting structural rearrangements significantly influence binding thermodynamics.
Researchers have developed a compact, all-in-one CRISPR-dCas9-based repressor platform delivered via AAV, effectively silencing gene expression relevant to neurodegenerative disorders, including Alzheimer's disease. The compact I-F2 system, smaller than the widely used SpCas9, was engineered to perform transcriptional activation by fusing various Cas proteins from the Cascade complex to an activation domain (VPR) or the transcription repression domains from MeCP2 and KRAB.

CRISPR screens

A CRISPR-based screen has revealed that the transcription factor ZNF740 plays a pivotal role in determining the sensitivity of acute myeloid leukaemia (AML) cells to venetoclax, a BCL-2 inhibitor. The study highlights ZNF740's regulation of the pro-apoptotic protein NOXA, which influences the stability of MCL-1, a critical factor in venetoclax resistance.
Researchers investigating triple-negative breast cancer (TNBC) identified key transcriptional enhancers and their non-coding enhancer RNAs (eRNAs) that drive aggressive tumour behaviour. Using CRISPR-interference assays and high-resolution Hi-C chromatin maps, they pinpointed super-enhancers linked to poor clinical outcomes. Disruption of one such super-enhancer, controlling the PODXL gene, significantly reduced TNBC cell proliferation, migration, and tumour growth in mouse models.

Detection

Researchers have integrated cascading CRISPR-Cas and nanozymes with electronic amplification in an organic photoelectrochemical transistor (OPECT) for enhanced miRNA detection. Using a CRISPR-Cas13a system and polyoxometalate nanozyme, this OPECT sensor achieves triple signal amplification, enabling sensitive detection of miRNA-21 with a linear range of 1 fM to 10 nM and a detection limit of 0.53 fM.
A novel diagnostic method for male infertility combines rolling circle transcription (RCT) with Cas13a techniques to detect piRNAs associated with infertility. This assay leverages RCT's amplification efficiency and Cas13a's RNase activities to create a highly sensitive and specific detection system. The method achieved a detection limit of 3.32 fM and demonstrated high accuracy and reproducibility in human serum samples, showing promise for clinical applications in diagnosing male infertility.

Reviews

Advances in delivery systems for CRISPR/Cas-mediated cancer treatment: a focus on viral vectors and extracellular vesicles. This review underscores the importance of optimized delivery mechanisms in realizing the full therapeutic potential of CRISPR/Cas technology in oncology.
CRISPR-Cas target recognition for sensing viral and cancer biomarkers. This review examines the potential of CRISPR-Cas systems for nucleic acid-based diagnostics, focusing on Cas9, Cas12, and Cas13 proteins.
CRISPR-Cas-based biosensors for cancer biomarkers detection. This review highlights the potential of CRISPR-Cas systems in noninvasive cancer diagnostics, focusing on their flexibility, sensitivity, and specificity.

Meetings

The 7th International Conference on CRISPR Technologies will take place October 14–16, 2024, at the Bahia Resort Hotel in San Diego, California. Organized by the Society for Biological Engineering (SBE) — a technical community of the American Institute of Chemical Engineers (AIChE) — the CRISPR conference unites the field's innovators and thought leaders, who will deliver academic and commercial perspectives on CRISPR-based techniques, highlighting the potential of genome engineering for applications in biotechnology, agriculture, biomedicine, and other fields.

Commentaries

In a recent paper, researchers argue that the $2.2 million cost per patient for Casgevy, the first approved CRISPR therapy, raises significant ethical concerns regarding equitable access and global health. Using Casgevy as a case study, they explore the factors contributing to its high price and the broader implications for future gene therapies. The authors advocate for urgent price reductions in CRISPR treatments to ensure justice and accessibility for patients with serious genetic diseases, highlighting the ethical imperative of making these life-saving therapies affordable on a global scale.
A Viewpoint on Biocompare.com highlights the evolution of CRISPR technology, emphasizing its shift from plasmid-based methods to advanced techniques like ribonucleoprotein (RNP) delivery, which offers more precise, transient editing with fewer off-target effects. Despite these advancements, CRISPR remains complex, prompting many researchers to collaborate with experts to ensure efficiency and consistency. Future developments will focus on increasing precision and expanding the CRISPR toolkit with innovations like base and prime editing.