An engineered exosome for delivering sgRNA:Cas9 ribonucleoprotein complex and genome editing in recipient cells

CRISPR-Cas9 is a versatile genome-editing technology that is a promising gene therapy tactic. However, the delivery of CRISPR-Cas9 is still a major obstacle to its broader clinical application. Here, we confirm that the components of CRISPR-Cas9-sgRNA and Cas9 protein-can be packaged into exosomes, where sgRNA and Cas9 protein exist as a sgRNA:Cas9 ribonucleoprotein complex. Although exosomal CRISPR-Cas9 components can be delivered into recipient cells, they are not adequate to abrogate the target gene in recipient cells. To solve this, we engineered a functionalized exosome (M-CRISPR-Cas9 exosome) that could encapsulate CRISPR-Cas9 components more efficiently. To improve the loading efficiency of Cas9 proteins into exosomes, we artificially engineered exosomes by fusing GFP and GFP nanobody with exosomal membrane protein CD63 and Cas9 protein, respectively. Therefore, Cas9 proteins could be captured selectively and efficiently loaded into exosomes due to the affinity of GFP-GFP nanobody rather than random loading. sgRNA and Cas9 protein exist as a complex in functionalized exosomes and can be delivered into recipient cells. To show the function of modified exosomes-delivered CRISPR-Cas9 components in recipient cells visually, we generated a reporter cell line (A549stop-DsRed) that produced a red fluorescent signal when the stop element was deleted by the sgRNA-guided endonuclease. Using A549stop-DsRed reporter cells, we showed that modified exosomes loaded with CRISPR-Cas9 components abrogated the target gene more efficiently in recipient cells. Our study reports an alternative tactic for CRISPR-Cas9 delivery.