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 t...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2022-04-01
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| Series: | Journal of Extracellular Vesicles |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/jev2.12196 |
| _version_ | 1818024491936120832 |
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| author | Joseph Andrew Whitley Sungjin Kim Lei Lou Chenming Ye Omar Awad Alsaidan Essilvo Sulejmani Jingwen Cai Ellison Gerona Desrochers Zanna Beharry Catherine Bowes Rickman Mikael Klingeborn Yutao Liu Zhong‐Ru Xie Houjian Cai |
| author_facet | Joseph Andrew Whitley Sungjin Kim Lei Lou Chenming Ye Omar Awad Alsaidan Essilvo Sulejmani Jingwen Cai Ellison Gerona Desrochers Zanna Beharry Catherine Bowes Rickman Mikael Klingeborn Yutao Liu Zhong‐Ru Xie Houjian Cai |
| author_sort | Joseph Andrew Whitley |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-10T04:01:04Z |
| format | Article |
| id | doaj.art-5004da086bee4b22b97e9a63aac1f15d |
| institution | Directory Open Access Journal |
| issn | 2001-3078 |
| language | English |
| last_indexed | 2024-12-10T04:01:04Z |
| publishDate | 2022-04-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Vesicles |
| spelling | doaj.art-5004da086bee4b22b97e9a63aac1f15d2022-12-22T02:02:58ZengWileyJournal of Extracellular Vesicles2001-30782022-04-01114n/an/a10.1002/jev2.12196Encapsulating Cas9 into extracellular vesicles by protein myristoylationJoseph Andrew Whitley0Sungjin Kim1Lei Lou2Chenming Ye3Omar Awad Alsaidan4Essilvo Sulejmani5Jingwen Cai6Ellison Gerona Desrochers7Zanna Beharry8Catherine Bowes Rickman9Mikael Klingeborn10Yutao Liu11Zhong‐Ru Xie12Houjian Cai13Department of Pharmaceutical and Biomedical Sciences College of Pharmacy University of Georgia Athens Georgia USADepartment of Pharmaceutical and Biomedical Sciences College of Pharmacy University of Georgia Athens Georgia USASchool of Electrical and Computer Engineering College of Engineering University of Georgia Athens Georgia USADepartment of Pharmaceutical and Biomedical Sciences College of Pharmacy University of Georgia Athens Georgia USADepartment of Pharmaceutical and Biomedical Sciences College of Pharmacy University of Georgia Athens Georgia USADepartment of Pharmaceutical and Biomedical Sciences College of Pharmacy University of Georgia Athens Georgia USADepartment of Cellular Biology and Anatomy Augusta University Augusta Georgia USASchool of Electrical and Computer Engineering College of Engineering University of Georgia Athens Georgia USADepartment of Chemical and Physical Sciences University of Virgin Islands St. Thomas Virgin IslandsDepartment of Ophthalmology Duke University Durham North Carolina USADepartment of Ophthalmology Duke University Durham North Carolina USADepartment of Cellular Biology and Anatomy Augusta University Augusta Georgia USASchool of Electrical and Computer Engineering College of Engineering University of Georgia Athens Georgia USADepartment of Pharmaceutical and Biomedical Sciences College of Pharmacy University of Georgia Athens Georgia USAAbstract 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.https://doi.org/10.1002/jev2.12196CRISPR/Cas9extracellular vesiclesmyristoylationSrc kinase |
| spellingShingle | Joseph Andrew Whitley Sungjin Kim Lei Lou Chenming Ye Omar Awad Alsaidan Essilvo Sulejmani Jingwen Cai Ellison Gerona Desrochers Zanna Beharry Catherine Bowes Rickman Mikael Klingeborn Yutao Liu Zhong‐Ru Xie Houjian Cai Encapsulating Cas9 into extracellular vesicles by protein myristoylation Journal of Extracellular Vesicles CRISPR/Cas9 extracellular vesicles myristoylation Src kinase |
| title | Encapsulating Cas9 into extracellular vesicles by protein myristoylation |
| title_full | Encapsulating Cas9 into extracellular vesicles by protein myristoylation |
| title_fullStr | Encapsulating Cas9 into extracellular vesicles by protein myristoylation |
| title_full_unstemmed | Encapsulating Cas9 into extracellular vesicles by protein myristoylation |
| title_short | Encapsulating Cas9 into extracellular vesicles by protein myristoylation |
| title_sort | encapsulating cas9 into extracellular vesicles by protein myristoylation |
| topic | CRISPR/Cas9 extracellular vesicles myristoylation Src kinase |
| url | https://doi.org/10.1002/jev2.12196 |
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