Encapsulating Cas9 into extracellular vesicles by protein myristoylation

Abstract

CRISPR/Cas9 genome editing is a very promising avenue for the treatment of a variety of genetic diseases. However, it is still very challenging to encapsulate CRISPR/Cas9 machinery for delivery. Protein N‐myristoylation is an irreversible co/post‐translational modification that results in the covalent attachment of the myristoyl‐group to the N‐terminus of a target protein. It serves as an anchor for a protein to associate with the cell membrane and determines its intracellular trafficking and activity. Extracellular vesicles (EVs) are secreted vesicles that mediate cell‐cell communication. In this study, we demonstrate that myristoylated proteins were preferentially encapsulated into EVs. The octapeptide derived from the leading sequence of the N‐terminus of Src kinase was a favourable substrate for N‐myristoyltransferase 1, the enzyme that catalyzes myristoylation. The fusion of the octapeptide onto the N‐terminus of Cas9 promoted the myristoylation and encapsulation of Cas9 into EVs. Encapsulation of Cas9 and sgRNA‐eGFP inside EVs was confirmed using protease digestion assays. Additionally, to increase the transfection potential, VSV‐G was introduced into the EVs. The encapsulated Cas9 in EVs accounted for 0.7% of total EV protein. Importantly, the EVs coated with VSV‐G encapsulating Cas9/sgRNA‐eGFP showed up to 42% eGFP knock out efficiency with limited off‐target effects in recipient cells. Our study provides a novel approach to encapsulate CRISPR/Cas9 protein and sgRNA into EVs. This strategy may open an effective avenue to utilize EVs as vehicles to deliver CRISPR/Cas9 for genome‐editing‐based gene therapy.

New Therapeutics for Extracellular Vesicles: Delivering CRISPR for Cancer Treatment

“…Ran et al produced a stable HEK293T cell line with the EGFP gene inserted into the genome. Upon treatment with CRISPR/Cas9-containing EVs, EGFP expression was significantly reduced, suggesting the function of EV-based DNA editing [ 114 ]. Another lab generated a reporting system in the lung carcinoma cell line A549, where a stop element was added in between the promoter and the DsRed coding sequence.…”

“…This modification added a long carbon chain to the glycine residue of the peptide, serving as a signal to the membrane localization. Intriguingly, 1.47% of total proteins in wild-type EVs were modified with the myristoyl group with unknown functions [ 114 ]. As expected, the myristoylated Cas9 was more likely than the native Cas9 to be sorted within EVs.…”

Advances in the Therapeutic Effects of Apoptotic Bodies on Systemic Diseases

“…Several studies have reported that the recombination of EVs can be achieved by modifying the donor cells [ 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 ]. In addition, to engineer the construction of EVs, multiple methods for therapeutic molecule loading can be used.…”

Advances in biomaterials for the treatment of retinoblastoma

“…342 Similarly, a more recent study highlighted that myristoylated proteins and the fusion of the octapeptide derived from Src kinase to the N-terminus of the Cas9 protein promotes its encapsulation into EVs. 343 Of more relevance to retinoblastoma, it has been shown that siRNA-loaded onto nanoparticles (targeting survivin) followed by adjuvant chemotherapy showed the capacity to drive tumor cell death in retinoblastoma in vitro. 344 A combination novel approach has been shown that the encapsulation of miR-181a and melphalan into about 170 nm lipid nanoparticle decreased retinoblastoma cell counts in an in vivo xenograft retinoblastoma rat model.…”

“…342 Similarly, a more recent study highlighted that myristoylated proteins and the fusion of the octapeptide derived from Src kinase to the N-terminus of the Cas9 protein promotes its encapsulation into EVs. 343…”