Lipid-encapsulated dual-cleaving endonuclease for DNA and gene editing
Methods to edit genes by administering a chimeric nuclease to a cell or organism without the use of a viral vector.
1. A nuclease comprising a modified Staphylococcus aureus Cas9 comprising the sequence of SEQ ID NO: 14.
2. The nuclease of claim 1 , wherein the modified Staphylococcus aureus Cas9 further comprises a histidine to alanine substitution at an amino acid corresponding to position 557 of SEQ ID NO: 13.
3. The nuclease of claim 1 , further comprising an Enterobacteria Phage T4 I-TevI nuclease domain.
4. The nuclease of claim 3 , wherein the I-TevI nuclease domain comprises the amino acid sequence set forth in SEQ ID NO: 6.
5. The nuclease of claim 4 , wherein the I-TevI nuclease domain comprises a substitution or deletion of the methionine at position 1 of SEQ. ID NO: 6.
6. The nuclease of claim 1 , further comprising a linker coupling the modified Staphylococcus aureus Cas9 and the I-TevI nuclease domain.
7. The nuclease of claim 6 , wherein the linker comprises an amino acid sequence set forth in any one or more of SEQ ID NO: 7 to SEQ ID NO 12.
8. The nuclease of claim 1 , bound to a guide RNA.
9. The nuclease of claim 8 , wherein the guide RNA comprises one or more bridged nucleic acids.
10. A chimeric nuclease comprising an I-TevI nuclease domain, a linker, a Staphylococcus aureus Cas9, and a guide RNA, wherein the Staphylococcus aureus Cas9 comprises the sequence of SEQ ID NO: 14.
11. The chimeric nuclease of claim 10 , wherein the Staphylococcus aureus Cas9 comprises an aspartic acid to glutamic acid substitution at an amino acid corresponding to position 10 of SEQ ID NO: 13.
12. The chimeric nuclease of claim 10 , wherein the I-TevI nuclease domain comprises the amino acid sequence set forth in SEQ ID NO: 6.
13. The chimeric nuclease of claim 12 , wherein the I-TevI nuclease domain comprises a substitution or deletion of the methionine at position 1 of SEQ NO: 6.
14. The chimeric nuclease of claim 10 , wherein the linker comprises an amino acid sequence set forth in any one or more of SEQ ID NO: 7 to SEQ ID NO 12.
15. A formulation comprising the chimeric nuclease of claim 11 and a lipid nanoparticle.
16. A method to genetically modify the genome of a cell, the method comprising: contacting the cell with the chimeric nuclease of claim 10 .
17. A nucleic acid encoding the nuclease of claim 1 .
18. A nucleic acid encoding the chimeric nuclease of claim 10 .