Delivery of CAS9 via ARRDC1-mediated microvesicles (ARMMs)
Methods, systems, compositions and strategies for the delivery of WW domain-containing fusion proteins into cells in vivo, ex vivo, or in vitro via ARMMs are provided. Methods, systems, compositions and strategies for the delivery of Cas9 proteins and/or Cas9 variants into cells in vivo, ex vivo, or in vitro via fusion to ARMM associated proteins (e.g., ARRDC1 or TSG101) are also provided.
1. An arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM), comprising:
(i) a lipid bilayer and an ARRDC1 protein, and
(ii) a cargo protein, wherein the cargo protein is linked to the ARRDC1 protein via one or more WW domains, and wherein the cargo protein is a Cas9 cargo protein.
2. The microvesicle of claim 1 , comprising an ARRDC1 protein, wherein the ARRDC1 protein comprises any one of the ARRDC1 amino acid sequences set forth in SEQ ID NOs: 15-17.
3. The microvesicle of claim 1 , wherein the Cas9 cargo protein is a nuclease active Cas9 cargo protein.
4. The microvesicle of claim 1 , wherein the Cas9 cargo protein is a nuclease inactive Cas9 (dCas9) cargo protein or a Cas9 nickase cargo protein.
5. The microvesicle of claim 1 , wherein the Cas9 cargo protein comprises an amino acid sequence that is at least 90% identical to a wild-type Cas9 protein.
6. The microvesicle of claim 1 , wherein the Cas9 cargo protein comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.
7. The microvesicle of claim 1 , wherein the Cas9 cargo protein comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.
8. The microvesicle of claim 1 , wherein the Cas9 cargo protein comprises at least one nuclear localization sequence (NLS).
9. The microvesicle of claim 1 , wherein the Cas9 cargo protein is bound to a guide RNA (gRNA).
10. The microvesicle of claim 9 , wherein the guide RNA is a single guide RNA (sgRNA).
11. The microvesicle of claim 1 , wherein the Cas 9 cargo protein is linked to the ARRDC1 protein via two WW domains.
12. The microvesicle of claim 11 , wherein the two WW domains plus the Cas9 cargo protein comprise the amino acid sequence set forth in SEQ ID NO: 65.
13. The microvesicle of claim 1 , wherein the Cas9 cargo protein is linked to the ARRDC1 protein via four WW domains.
14. The microvesicle of claim 13 , wherein the four WW domains plus the Cas9 cargo protein comprise the amino acid sequence set forth in SEQ ID NO: 66.
15. The microvesicle of claim 1 , wherein the ARRDC1 protein comprises a HA tag.
16. The microvesicle of claim 1 , wherein the ARRDC1 protein comprises a GFP.
17. The microvesicle of claim 1 , wherein one or more of the WW domains comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64.