Modulating binding affinity of aptamer-based loading constructs enhances extracellular vesicle-mediated CRISPR/Cas9 delivery

The CRISPR/Cas9 toolbox consists of modular nucleases that can be employed to efficiently modify genomic sequences with high specificity. However, delivery of the large Cas9-sgRNA ribonucleoprotein (RNP) complexes remains challenging due to their immunogenicity, size, and overall negative charge. An approach to overcome these limitations is the use of extracellular vesicles (EVs) as intracellular delivery vehicles. EVs exhibit the natural ability to carry and deliver RNA and proteins across biological barriers, and can be engineered to load and deliver a variety of biotherapeutic molecules. Previous studies have shown that efficient EV-mediated cargo delivery does not only require active loading strategies, but also benefits from strategies to release cargo from the EV membrane. Here, we load Cas9 RNP complexes into EVs by expressing sgRNAs containing MS2 aptamers (MS2-sgRNAs), alongside Cas9 and a fusion protein of CD63 and tandem MS2 coat proteins (MCPs). We demonstrate that efficient Cas9 RNP delivery can also be facilitated by modulating the binding affinity between MS2 aptamers and the MCPs. To study the effect of altering the binding affinity between the MS2 hairpin and the MCP on Cas9 RNP delivery, various mutations affecting the binding affinity were made in both the interacting MS2-hairpin and the RNA-binding domain of the MCPs. Comparing Cas9 RNP delivery of the modulated MS2-sgRNAs revealed that adapting binding affinity highly affects functional RNP delivery. Mutations resulting in high affinity did not facilitate efficient RNP delivery unless combined with a photo-inducible release strategy, showing that cargo release was a limiting factor in RNP delivery. Mutations that decreased affinity resolved this issue, resulting in Cas9 RNP delivery without the requirement of additional release strategies. However, further decreasing affinity resulted in decreased Cas9 gene-editing efficiency due to decreased levels of Cas9 RNP loading into EVs. A similar effect on functional delivery was seen after modification of the RNA-binding domain of the MCPs. Our results demonstrate that EVs are capable of functional Cas9-sgRNA complex delivery, and that modulation of binding affinity can be used to increase efficient functional delivery with non-covalent loading constructs, without the need for additional engineering strategies for cargo release.