Hegeman Charlotte V, Elsharkasy Omnia M, Driedonks Tom A P, Friesen Kate R J, Vader Pieter, de Jong Olivier G
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, the Netherlands.
CDL Research, University Medical Center Utrecht, Utrecht, the Netherlands.
J Control Release. 2025 Aug 10;384:113853. doi: 10.1016/j.jconrel.2025.113853. Epub 2025 May 18.
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.
CRISPR/Cas9工具盒由模块化核酸酶组成，可用于高效且高度特异性地修饰基因组序列。然而，由于大型Cas9-sgRNA核糖核蛋白（RNP）复合物具有免疫原性、尺寸较大且整体带负电荷，其递送仍然具有挑战性。克服这些限制的一种方法是使用细胞外囊泡（EV）作为细胞内递送载体。EV具有携带和递送RNA及蛋白质跨越生物屏障的天然能力，并且可以进行工程改造以装载和递送多种生物治疗分子。先前的研究表明，高效的EV介导的货物递送不仅需要主动装载策略，还受益于从EV膜释放货物的策略。在这里，我们通过表达含有MS2适体的sgRNA（MS2-sgRNA），同时表达Cas9以及CD63与串联MS2外壳蛋白（MCP）的融合蛋白，将Cas9 RNP复合物装载到EV中。我们证明，通过调节MS2适体与MCP之间的结合亲和力，也可以促进高效的Cas9 RNP递送。为了研究改变MS2发夹与MCP之间的结合亲和力对Cas9 RNP递送的影响，在相互作用的MS2发夹和MCP的RNA结合结构域中都进行了各种影响结合亲和力的突变。比较经调节的MS2-sgRNA的Cas9 RNP递送情况发现，调整结合亲和力对功能性RNP递送有很大影响。导致高亲和力的突变除非与光诱导释放策略相结合，否则不会促进有效的RNP递送，这表明货物释放是RNP递送中的一个限制因素。降低亲和力的突变解决了这个问题，使得Cas9 RNP递送无需额外的释放策略。然而，进一步降低亲和力会导致Cas9基因编辑效率降低，因为进入EV的Cas9 RNP水平下降。对MCP的RNA结合结构域进行修饰后，对功能性递送也观察到了类似的影响。我们的结果表明，EV能够进行功能性的Cas9-sgRNA复合物递送，并且结合亲和力的调节可用于通过非共价装载构建体提高有效功能性递送，而无需额外的货物释放工程策略。
