Mastrobattista, E. | bioRxiv Science

bioRxiv2024

A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

CRISPR-Cas9 gene editing technology offers the potential to permanently repair genes containing pathological mutations. However, efficient intracellular delivery of the Cas9 ribonucleoprotein complex remains one of the major hurdles in its therapeutic application. Extracellular vesicles (EVs) are biological nanosized membrane vesicles released by cells, that play an important role in intercellular communication. Due to their innate capability of intercellular transfer of proteins, RNA, and various other biological cargos, EVs have emerged as a novel promising strategy for the delivery of macromolecular biotherapeutics, including CRISPR-Cas9 ribonucleoproteins. Here, we present a versatile, modular strategy for the loading and delivery of Cas9. We leverage the high affinity binding of MS2 coat proteins (MCPs) fused to EV-enriched proteins to MS2 aptamers incorporated into single guide RNAs (sgRNAs), in combination with a UV-activated photocleavable linker domain, PhoCl. Combined with the Vesicular stomatitis virus G (VSV-G) protein this modular platform enables efficient loading and subsequent delivery of the Cas9 ribonucleoprotein complex, which shows critical dependence on the incorporation and activation of the photocleavable linker domain. As this approach does not require any direct fusion of Cas9 to EV-enriched proteins, we demonstrate that Cas9 can readily be exchanged for other variants, including transcriptional activator dCas9-VPR and adenine base editor ABE8e, as confirmed by various sensitive fluorescent reporter assays. Taken together, we describe a robust and modular strategy for successful Cas9 delivery, which can be applied for CRISPR-Cas9-based genetic engineering as well as transcriptional regulation, underlining the potential of EV-mediated strategies for the treatment of genetic diseases.

molecular biology↗

bioRxiv2023

Anti-cancer compound screening identifies Aurora Kinase A inhibition as a means to favor CRISPR/Cas9 gene correction over knock-out

CRISPR gene therapy holds the potential to cure a variety of genetic diseases by targeting causative mutations and introducing double stranded DNA breaks, subsequently allowing the host DNA repair mechanisms to introduce mutations. One option to introduce precise gene corrections is via the homology-directed repair (HDR) pathway. HDR can introduce a range of desired mutations dictated by a DNA template which holds a corrected DNA sequence which is written into the targeted gene. The problem in utilizing this pathway is that CRISPR-induced double stranded DNA breaks are repaired more often through the non-homologous end joining (NHEJ) pathway, which does not use a designed template and introduces random DNA damage in the form of insertions and deletions at the cut site. Since HDR activation depends on many interconnected processes in the cell, we aimed to screen a small library of drug compounds in clinical use or clinical development for cancer, to steer the DNA repair process towards preferential HDR activation. We included compounds in our screen based on three relevant mechanisms in CRISPR gene editing: the cell cycle, DNA repair processing and chromosomal packing. We included forty compounds, based on these criteria, screened their toxicity and dosed them in sub-toxic concentrations in cells during genome editing. Of these forty compounds we identified nine potential hits to have an effect on preferential activation of the HDR pathway over NHEJ. Alisertib, rucaparib and belinostat revealed a significant and major effect on gene editing pathway selection in further validation. Alisertib, an Aurora kinase A inhibitor, showed a particularly strong effect towards improving HDR over NHEJ. We subsequently investigated this effect at the genetic level and in a murine hepatoma cell line, which corroborated the initial findings. Alisertib led to an over 4-fold increase in preferential gene correction over gene knock-out, at a dose of 0.3 micromolar. However, the observations that Aurora kinase A inhibitors show considerable cytotoxicity (<50% cell viability) and can induce morphological changes at this concentration pose a limitation for the direct use of these inhibitors as HDR enhancers. However these findings do implicate that the pathways mediated by Aurora kinase A strongly influence HDR outcomes, which warrants further investigation into the downstream pathways driving this effect.

pharmacology and toxicology↗