Begoña Sot

The efficient and safe intracellular delivery of CRISPR ribonucleoproteins (RNPs) remains a major challenge for the development of genome editing strategies. Magnetic iron oxide nanoparticles (MNPs) offer an attractive and biocompatible delivery platform. However, their use for the delivery of functional CRISPR RNPs remains comparatively underexplored. Here, we developed carboxymethyl dextran (CMD)-coated MNPs as non-viral carriers for Cas9 and Cas12a RNPs. Nanocomplexes were formed through electrostatic interactions, without the need for chemical coupling reagents or additional surface functionalization. Both RNPs efficiently associated with MNPs, and the resulting nanocomplexes exhibited stable physicochemical properties and retained their cargo in serum-supplemented culture medium for up to 24 hours. Cellular uptake was confirmed by microscopy, while biocompatibility assays revealed no overt cytotoxicity. Functional genome editing was demonstrated at reporter and endogenous gene loci in HEK-293T cells, as well as in primary human dermal fibroblasts. Finally, we explored the therapeutic potential of this platform for recessive dystrophic epidermolysis bullosa (RDEB) by targeting COL7A1 exon 80, with the aim of removing the prevalent c.6527insC mutation while preserving the reading frame. Cas9 RNP nanocomplexes enabled exon 80 deletion. These findings establish CMD-coated MNPs as a versatile platform for the delivery of Cas9 and Cas12a RNPs and support further investigation of magnetic nanoparticle-mediated genome editing for inherited skin disorders.

Rare diseases are serious and often chronic conditions that affect a small number of individuals. However, with over 7,000 rare diseases identified, their cumulative global numbers and impact are substantial. A considerable proportion of these conditions is caused by genetic abnormalities. Among these, monogenic disorders are of particular relevance, as they are caused by mutations in specific genes. The development of gene therapy, and more specifically, gene editing, offers innovative approaches to treat these rare diseases. A significant challenge associated with the implementation of such strategies concerns the delivery of gene editing tools. Nonviral vectors based on nanomaterials have demonstrated considerable potential as promising alternatives to viral vectors, thereby overcoming their disadvantages. The biocompatibility and tunability of nanoparticles, along with their potential capacity to target diverse tissues, positions them as a promising therapeutic approach for the treatment of a wide range of organ-specific rare diseases. Here, we review current progress in the development and evaluation of novel nanomedicine strategies for gene editing in rare diseases, highlighting new gene editing approaches, delivery systems, and potential targets.

Uveal melanoma (UM) is a rare ocular tumor characterized by high metastasis risk and poor prognosis. The in-depth characterization of UM's molecular profile is critical for better disease classification and prognosis. Furthermore, the development of detection tools to monitor UM evolution upon treatment is of great interest for designing optimal therapeutic strategies. However, commonly used techniques, such as ddPCR or NGS, are costly, and they involve sophisticated equipment and complex experimental design. The development of alternative sensing methods that are fast, simple, and inexpensive would be of great benefit to improve UM's diagnosis and management, especially when combined with liquid biopsy. Samples from liquid biopsy can be obtained with minimal invasiveness, and the detection of circulating tumor DNA (ctDNA) in UM patients' plasma has proven useful for the diagnosis of metastasis, prognosis prediction, and disease monitoring. In this context, CRISPR/Cas12a-derived molecular sensors, thanks to their high specificity and sensitivity and their potential for point of care diagnosis, are particularly interesting. Here, we developed a CRISPR/Cas12a-based approach for the specific detection of the UM-related mutation GNAQ Q209P that relies on the design of highly specific crRNAs. Coupled with allele-specific PCR, it constitutes a sensitive platform for liquid biopsy detection, capable of sensing GNAQ Q209P in plasma samples with a low ctDNA concentration and fractional abundance. Finally, our method was validated using plasma samples from metastatic UM patients.

CRANAD‐2 is a fluorogenic curcumin derivative used for near‐infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD‐2 in two super‐resolution imaging techniques, namely stimulated emission depletion (STED) and single‐molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD‐2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45–55 nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super‐resolved image. The good performance and versatility of CRANAD‐2 provides a powerful tool for near‐infrared nanoscopic imaging of amyloids in vitro and in vivo.

The versatile clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has emerged as a promising technology for therapy and molecular diagnosis. It is especially suited for overcoming viral infections outbreaks, since their effective control relies on an efficient treatment, but also on a fast diagnosis to prevent disease dissemination. The CRISPR toolbox offers DNA‐ and RNA‐targeting nucleases that constitute dual weapons against viruses. They allow both the manipulation of viral and host genomes for therapeutic purposes and the detection of viral nucleic acids in “Point of Care” sensor devices. Here, we thoroughly review recent advances in the use of the CRISPR/Cas system for the treatment and diagnosis of viral deleterious infections such as HIV or SARS‐CoV‐2, examining their strengths and limitations. We describe the main points to consider when designing CRISPR antiviral strategies and the scientific efforts to develop more sensitive CRISPR‐based viral detectors. Finally, we discuss future prospects to improve both applications. Also see the video abstract here: https://www.youtube.com/watch?v=C0z1dLpJWl4

We report the use of the amyloid probe Thioflavin T (ThT) as a specific and exchangeable fluorophore for stimulated emission depletion (STED) super-resolution imaging of amyloid fibers.

α-Synuclein is an intrinsically disordered protein whose aggregation in form of amyloid fibres is directly implicated in Parkinson’s disease and other neurological disorders. α-Synuclein is composed of three different regions. The central region (61-95), called NAC, is responsible for protein fibrillation. The N-terminal region (1-61) has some helical propensity and can be divided in H1 (1-31) and H2 (32-61), while the highly acidic C-terminal region (96-140) is completely disordered. It has been postulated that the acidic character of the C-terminus, as well as the interaction between the soluble N- and C- terminal parts, protects the NAC region from fibrillation. In consequence, N- and C- terminal deletions increase α Synuclein fibrillation. Both N- and C- terminal truncations are common in synucleinopathies, but despite their clinical relevance, to date there are no systematic and exhaustive studies that quantify the effect of these truncations in fibre nucleation and elongation. In this work, we measured both nucleation and fibrillation elongation kinetics in order to study the influence of N- and C- terminal deletions, including the simultaneous deletion of several regions, in α-Synuclein fibrillation. We also tested whether the fibrillation prone mutation A53T had an additional effect when combined with truncations. Furthermore, our cross-seeding experiments showed that the deletions studied induce changes in fibre morphology. Our results unravel then the role of the different α-Synuclein regions and the A53T mutation in the nucleation and elongation of amyloid fibres.