Engineered nucleic acid-targeting nucleic acids
The present disclosure provides engineered Class 2 CRISPR-Cas-associated discontinuous first-stem nucleic-acid targeting nucleic acids, nucleoprotein complexes comprising these nucleic acids, and compositions thereof. Nucleic acid sequences encoding the Class 2 CRISPR-Cas-associated discontinuous first-stem nucleic-acid targeting nucleic acids, as well as expression cassettes, vectors and cells comprising such nucleic acid sequences, are described. Also, methods are disclosed for making and using the Class 2 CRISPR-Cas-associated discontinuous first-stem nucleic-acid targeting nucleic acids, nucleoprotein complexes comprising such nucleic acids, and compositions thereof.
1. A Class 2 Type II CRISPR-Cas9-associated composition, comprising:
(i) a first Class 2 Type II CRISPR-Cas9-associated discontinuous first-stem single-strand polynucleotide (dfs1-PN) comprising, in a 5′ to 3′ direction,
a fragment nucleotide sequence 1 comprising a 5′ terminus of the dfs1-PN and at least 2 nucleotides,
a bulge element nucleotide sequence II,
an upper stem element nucleotide sequence II,
a first linker nucleotide sequence,
an upper stem element nucleotide sequence I,
a bulge element nucleotide sequence I, and
a lower stem element nucleotide sequence I,
a second linker nucleotide sequence,
a nexus nucleotide sequence,
a third linker nucleotide sequence, and
a 3′ hairpin element; and
(ii) a second Class 2 Type II CRISPR-Cas9-associated discontinuous first-stem single-strand polynucleotide (dfs2-PN) comprising, in a 5′ to 3′ direction,
a nucleic acid target binding sequence, and
a fragment nucleotide sequence 2 comprising at least 2 nucleotides and a 3′ terminus of the dfs2-PN;
wherein the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II base-pair hydrogen bond to form an upper stem element;
wherein the bulge element nucleotide sequence I and the bulge element nucleotide sequence II form a bulge element; and
wherein 2 nucleotides of the fragment nucleotide sequence 1 form 2 pairs of hydrogen-bonded nucleotides with the lower stem element nucleotide sequence I and 2 nucleotides of the fragment nucleotide sequence 2 form 2 pairs of hydrogen-bonded nucleotides with the lower stem element nucleotide sequence I to form a lower stem element;
or
(i) a first Class 2 Type II CRISPR-Cas9-associated discontinuous first-stem single-strand polynucleotide (dfs1-PN) comprising, in a 5′ to 3′ direction,
a fragment nucleotide sequence 1 comprising a 5′ terminus of the dfs1-PN and at least 2 nucleotides,
a first linker nucleotide sequence,
a nexus nucleotide sequence,
a second linker nucleotide sequence, and
a 3′ hairpin element; and
(ii) a second Class 2 Type II CRISPR-Cas9-associated discontinuous first-stem single-strand polynucleotide (dfs2-PN) comprising, in a 5′ to 3′ direction,
a nucleic acid target binding sequence,
a lower stem element nucleotide sequence II,
a bulge element nucleotide sequence II,
an upper stem element nucleotide sequence II,
a third linker nucleotide sequence,
an upper stem element nucleotide sequence I,
a bulge element nucleotide sequence I, and
a fragment nucleotide sequence 2 comprising at least 2 nucleotides and a 3′ terminus of the dfs2-PN;
wherein the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II base-pair hydrogen bond to form an upper stem element;
wherein the bulge element nucleotide sequence I and the bulge element nucleotide sequence II form a bulge element; and
wherein 2 nucleotides of the fragment nucleotide sequence 1 form 2 pairs of hydrogen-bonded nucleotides with the lower stem element nucleotide sequence II and 2 nucleotides of the fragment nucleotide sequence 2 form 2 pairs of hydrogen-bonded nucleotides with the lower stem element nucleotide sequence II to form a lower stem element.
2. The composition of claim 1 , wherein the lower stem nucleotide sequence I is up to 10 nucleotides.
3. The composition of claim 1 , wherein the lower stem nucleotide sequence I is 9 nucleotides, the bulge element nucleotide sequence I is 3 nucleotides, the bulge element nucleotide sequence II is 1 nucleotide, and the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II are each between 3 and 20 nucleotides.
4. The composition of claim 1 , wherein the lower stem nucleotide sequence I is 6 nucleotides, the bulge element nucleotide sequence I is 4 nucleotides, the bulge element nucleotide sequence II is 2 nucleotides, and the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II are each between 4 and 20 nucleotides.
5. The composition of claim 1 , wherein the upper stem nucleotide sequence I and the upper stem element nucleotide sequence II are each between 2 and 22 nucleotides.
6. The composition of claim 1 , wherein the dfs1-PN further comprises an additional hairpin element 3′ of the 3′ hairpin element.
7. The composition of claim 1 , wherein dfs1-PN comprises DNA, RNA, or DNA and RNA, and dfs2 PN comprises DNA, RNA, or DNA and RNA.
8. The composition of claim 1 , wherein dfs1-PN or dfs2-PN comprises a thiol moiety.
9. The composition of claim 1 , wherein a pair of the 2 pairs of hydrogen-bonded nucleotides at the 5′ terminus of the dfs1-PN is Watson-Crick-hydrogen-bonded or wobble-hydrogen-bonded; and wherein a pair of the 2 pairs of hydrogen-bonded nucleotides at the 3′ terminus of the dfs2-PN is Watson-Crick-hydrogen-bonded or wobble-hydrogen-bonded.
10. The composition of claim 9 , wherein the pair of hydrogen-bonded nucleotides at the 3′ terminus of the dfs2-PN is Watson-Crick-hydrogen-bonded.
11. A nucleoprotein composition, comprising:
the Class 2 Type II CRISPR-Cas9-associated composition of claim 1 ; and
a Cas9 protein.
12. The nucleoprotein composition of claim 11 , wherein the composition is in a complex with the Cas9 protein.
13. The nucleoprotein composition of claim 11 , wherein the Cas9 protein is enzymatically inactive.
14. A kit, comprising:
the Class 2 Type II CRISPR-Cas9-associated composition of claim 1 ; and
a buffer.
15. The kit of claim 14 , further comprising a Cas9 protein or a nucleotide sequence encoding a Cas9 protein.
16. The composition of claim 1 , wherein the lower stem element nucleotide sequence II is up to 10 nucleotides.
17. The composition of claim 1 , wherein the lower stem element nucleotide sequence II is 9 nucleotides, the bulge element nucleotide sequence I is 3 nucleotides, the bulge element nucleotide sequence II is 1 nucleotide, and the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II are each between 3 and 20 nucleotides.
18. The composition of claim 1 , wherein the lower stem element nucleotide sequence II is 6 nucleotides, the bulge element nucleotide sequence I is 4 nucleotides, the bulge element nucleotide sequence II is 2 nucleotides, and the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II are each between 4 and 20 nucleotides.
19. The composition of claim 1 , wherein dfs1-PN and dfs2-PN each comprise a thiol moiety.
20. The composition of claim 9 , wherein the pair of hydrogen-bonded nucleotides at the 5′ terminus of the dfs1-PN is Watson-Crick-hydrogen-bonded.