Methods and compositions for using argonaute to modify a single stranded target nucleic acid
The present disclosure provides compositions, kits, genetically modified cells, non-human transgenic organisms, and methods for binding and/or cleaving a single stranded target nucleic acid. A method of cleaving includes contacting a single stranded target nucleic acid with (e.g., introducing into a cell) a subject argonaute (Ago) polypeptide and a guide RNA (e.g., having a 5′-OH). In some embodiments, a subject Ago polypeptide includes an amino acid sequence having 70% or more sequence identity with amino acids 282-430 and/or 431-639 of the Marinitoga piezophila argonaute (MpAgo) protein set forth in SEQ ID NO: 1. The present disclosure provides variant Ago polypeptides; and methods of use of same.
1. A method of binding a single stranded target nucleic acid, the method comprising:
contacting the single stranded target nucleic acid with:
(i) an argonaute (Ago) polypeptide comprising (a) an amino-terminal domain (N-domain), (b) a PIWI/Argonaute/Zwille (PAZ) domain, (c) a MID domain, and (d) a PIWI domain,
wherein the MID domain comprises an amino acid sequence having 80% or more sequence identity with amino acids 282-430 of the wild type Marinitoga piezophila argonaute (MpAgo) amino acid sequence set forth in SEQ ID NO: 1, and/or the PIWI domain comprises an amino acid sequence having 80% or more sequence identity with amino acids 431-639 of the amino acid sequence set forth in SEQ ID NO: 1,
wherein the Ago polypeptide is a mutant MpAgo polypeptide that, compared to the wild type MpAgo protein set forth in SEQ ID NO: 1, comprises an amino acid sequence having one or more mutations and exhibits reduced nuclease activity; and
(ii) a guide RNA comprising a targeting nucleotide sequence of 15 to 35 nucleotides (nt) in length that is complementary to a target sequence of the target nucleic acid.
2. The method according to claim 1 , wherein the guide RNA comprises a 5′-OH.
3. The method according to claim 1 , wherein the length of the guide RNA is in a range of from 15 to 30 nucleotides.
4. The method according to claim 1 , wherein the length of the targeting nucleotide sequence of the guide RNA is in a range of from 15 to 30 nucleotides.
5. The method according to claim 1 , wherein the targeting nucleotide sequence of the guide RNA has 70% or more complementarity to the target sequence of the target nucleic acid over 15 or more contiguous nucleotides.
6. The method according to claim 1 , wherein the single stranded target nucleic acid is inside of a cell.
7. The method according to claim 6 , wherein the cell is a prokaryotic cell.
8. The method according to claim 6 , wherein the cell is a eukaryotic cell.
9. The method according to claim 6 , wherein contacting the target nucleic acid with the Ago polypeptide comprises introducing the Ago polypeptide into the cell, or introducing into the cell a nucleic acid encoding the Ago polypeptide.
10. The method according to claim 1 , wherein the mutant MpAgo polypeptide comprises an amino acid sequence having a substitution or deletion in one or more amino acid positions selected from: D446, E482, D516, and N624 compared to the wild type MpAgo protein set forth in SEQ ID NO: 1.
11. A recombinant expression vector comprising a nucleotide sequence that encodes:
an argonaute (Ago) polypeptide comprising (a) an amino-terminal domain (N-domain), (b) a PIWI/Argonaute/Zwille (PAZ) domain, (c) a MID domain, and (d) a PIWI domain,
wherein the MID domain comprises an amino acid sequence having 80% or more sequence identity with amino acids 282-430 of the wild type Marinitoga piezophila argonaute (MpAgo) amino acid sequence set forth in SEQ ID NO: 1, and/or the PIWI domain comprises an amino acid sequence having 70% or more sequence identity with amino acids 431-639 of the amino acid sequence set forth in SEQ ID NO: 1.
12. The recombinant expression vector of claim 11 , wherein the Ago polypeptide is:
(i) a mutant MpAgo polypeptide that, compared to the wild type MpAgo protein set forth in SEQ ID NO: 1, comprises an amino acid sequence having one or more mutations and exhibits reduced nuclease activity; or
(ii) a chimeric MpAgo polypeptide comprising a fusion partner having an amino acid sequence that provides for a modification of the target nucleic acid other than cleavage.
13. The recombinant expression vector of claim 11 , further comprising a nucleotide sequence that encodes a guide RNA that comprises a targeting nucleotide sequence of 15 to 35 nucleotides (nt) in length that is complementary to a target sequence of a single stranded target nucleic acid.
14. The recombinant expression vector of claim 13 , wherein the guide RNA comprises a 5′-OH.
15. A method of modifying a single stranded target nucleic acid, the method comprising:
contacting the single stranded target nucleic acid with:
A) a fusion polypeptide comprising:
a) an argonaute (Ago) polypeptide comprising: (i) an amino-terminal domain (N-domain), (ii) a PIWI/Argonante/Zwille (PAZ) domain, (iii) a MID domain, and (iv) a PIWI domain,
wherein the MID domain comprises an amino acid sequence having 80% or more sequence identity with amino acids 282-430 of the wild type Marinitoga piezophila argonaute (MpAgo) amino acid sequence set forth in SEQ ID NO: 1, and/or the PIWI domain comprises an amino acid sequence having 80% or more sequence identity with amino acids 431-639 of the amino acid sequence set forth in SEQ ID NO: 1; and
b) a fusion partner having an amino acid sequence that provides for a modification of the target nucleic acid other than cleavage; and
B) a guide RNA comprising a targeting nucleotide sequence of 15 to 35 nucleotides (nt) in length that is complementary to a target sequence of the target nucleic acid.
16. The method according to claim 15 , wherein the guide RNA comprises a 5′-OH.
17. The method according to claim 15 , wherein the length of the guide RNA is in a range of from 15 to 30 nucleotides.
18. The method according to claim 15 , wherein the length of the targeting nucleotide sequence of the guide RNA is in a range of from 15 to 30 nucleotides.
19. The method according to claim 15 , wherein the targeting nucleotide sequence of the guide RNA has 70% or more complementarity to the target sequence of the target nucleic acid over 15 or more contiguous nucleotides.
20. The method according to claim 15 , wherein the single stranded target nucleic acid is inside of a cell.
21. The method according to claim 20 , wherein the cell is a prokaryotic cell.
22. The method according to claim 20 , wherein the cell is a eukaryotic cell.
23. The method according to claim 20 , wherein contacting the target nucleic acid with the Ago polypeptide comprises introducing the Ago polypeptide into the cell, or introducing into the cell a nucleic acid encoding the Ago polypeptide.
24. A kit, comprising:
an argonaute (Ago) polypeptide, or a nucleic acid encoding the same, wherein the Ago polypeptide comprises (a) an amino-terminal domain (N-domain), (b) a PIWI/Argonaute/Zwille (PAZ) domain, (c) a MID domain, and (d) a PIWI domain,
wherein the MID domain comprises an amino acid sequence having 80% or more sequence identity with amino acids 282-430 of the wild type Marinitoga piezophila argonaute (MpAgo) amino acid sequence set forth in SEQ ID NO: 1, and/or the PIWI domain comprises an amino acid sequence having 70% or more sequence identity with amino acids 431-639 of the amino acid sequence set forth in SEQ ID NO: 1,
wherein the Ago polypeptide is a mutant MpAgo polypeptide that, compared to the wild type MpAgo protein set forth in SEQ ID NO: 1, comprises an amino acid sequence having one or more mutations and exhibits reduced nuclease activity.
25. The kit of claim 24 , wherein the guide RNA comprises a 5′-OH.
26. The kit of claim 25 , further comprising a 5′-OH generating nuclease or nucleic acid encoding the same.
27. The kit of claim 24 , further comprising a nucleotide sequence that encodes a guide RNA that comprises a targeting nucleotide sequence of 15 to 35 nucleotides (nt) in length that is complementary to a target sequence of a single stranded target nucleic acid.
28. A kit, comprising:
a fusion polypeptide comprising:
a) an argonaute (Ago) polypeptide comprising: (i) an amino-terminal domain (N-domain), (ii) a PIWI/Argonaute/Zwille (PAZ) domain, (iii) a MID domain, and (iv) a PIWI domain,
wherein the MID domain comprises an amino acid sequence having 80% or more sequence identity with amino acids 282-430 of the wild type Marinitoga piezophila argonaute (MpAgo) amino acid sequence set forth in SEQ ID NO: 1, and/or the PIWI domain comprises an amino acid sequence having 80% or more sequence identity with amino acids 431-639 of the amino acid sequence set forth in SEQ ID NO: 1; and
b) a fusion partner having an amino acid sequence that provides for a modification of the target nucleic acid other than cleavage.
29. The kit of claim 28 , wherein the guide RNA comprises a 5′-OH.
30. The kit of claim 29 , further comprising a 5′-OH generating nuclease or nucleic acid encoding the same.
31. The kit of claim 28 , further comprising a nucleotide sequence that encodes a guide RNA that comprises a targeting nucleotide sequence of 15 to 35 nucleotides (nt) in length that is complementary to a target sequence of a single stranded target nucleic acid.