CRISPR-Cas based system for targeting single-stranded sequences
The present invention relates to a CRISPR-Cas based system for targeting nucleic acid sequences. In part, the invention relates to synthetic guiding components for targeting single-stranded sequences, as well as design principles for constructing such components. Also described herein are methods of employing such components, e.g., to repress or activate a desired target within the subject.
1. A method of conducting a cleavage assay, the method comprising:
incubating a synthetic guiding component with a nuclease and a single-stranded target sequence, the synthetic guiding component including a targeting portion configured to bind and/or cleave the single-stranded target sequence; and
cleaving the single-stranded target sequence without a short DNA oligomer containing a proto-spacer adjacent motif (PAM) sequence (PAMmer);
wherein the synthetic guiding component comprises a structure having the formula (I):
W—X—Y-L-Z or a salt thereof, wherein:
W is an optional third portion comprising a nucleic acid sequence of from about 1 to 20 nucleic acids;
X is the targeting portion comprising a nucleic acid sequence configured to bind to a target site of the single-stranded target sequence;
Y is a first portion comprising a nucleic acid sequence configured to interact with a nuclease configured to cleave the single-stranded target sequence;
L is a linker; and
Z is a second portion comprising a nucleic acid sequence configured to interact with the nuclease and the first portion;
wherein the nuclease is a SauCas9 protein.
2. The method of claim 1 , wherein:
Y comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:150-155 or a complement of any of these; and
Z comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:162-178 or a complement of any of these.
3. The method of claim 1 , wherein:
L comprises a bond or a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:156-161, 187-191, 226-231, and 265-269, or a complement of any of these.
4. The method of claim 1 , wherein W and/or Y comprises one or more modified nucleic acids or bulges.
5. The method of claim 1 , wherein the single stranded target sequence recited for X is a single-stranded human mRNA target sequence or a single-stranded pathogen target sequence.
6. The method of claim 5 , X has a length of from about 15 to about 30 nucleotides.
7. The method of claim 1 , wherein the single-stranded target sequence is a single-stranded ribonucleic acid sequence.
8. The method of claim 1 , wherein the nuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:100, 101, and 110.
9. The method of claim 1 , wherein:
Y comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:179-186, or a complement of any of these, or a fragment thereof;
L is a bond or comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:187-191, or a complement of any of these, or a fragment thereof; and
Z comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:192-212, or a complement of any of these, or a fragment thereof.
10. The method of claim 1 , wherein W has a length of from 0 to about 20 nucleotides, X has a length of from about 10 to about 30 nucleotides, Y has a length of from about 10 to about 40 nucleotides, L has a length of from 0 to about 10 nucleotides, and Z has a length of from about 10 to about 90 nucleotides.
11. The method of claim 1 , wherein the structure has a length of from about 100 to about 200 nucleotides.
12. The method of claim 1 , further comprising detecting a label on the synthetic guiding component or a reporter on a nucleic acid substrate, wherein a detectable signal is provided upon recognition of the single-stranded target sequence by the synthetic guiding component.
13. The method of claim 12 , wherein the nuclease comprises an amino acid sequence corresponding to SEQ ID NOs: 100 or 101.
14. The method of claim 13 , wherein the nuclease comprises an amino acid sequence corresponding to SEQ ID NO: 101.
15. The method of claim 13 , wherein the nuclease comprises an amino acid sequence corresponding to SEQ ID NO: 100.
16. The method of claim 1 , wherein the single-stranded target sequence comprises an RNA sequence of a virus, the virus having a lifecycle consisting of solely RNA molecules.
17. A method of conducting a cleavage assay, the method comprising:
incubating a synthetic guiding component with a nuclease and a single-stranded target sequence, the synthetic guiding component including a targeting portion configured to bind and/or cleave the single-stranded target sequence; and
cleaving the single-stranded target sequence without a short DNA oligomer containing a proto-spacer adjacent motif (PAM) sequence (PAMmer);
wherein the synthetic guiding component comprises a structure having the formula (I):
W—X—Y-L-Z or a salt thereof,
wherein:
W is an optional third portion comprising a nucleic acid sequence of from about 1 to 20 nucleic acids;
X is the targeting portion comprising a nucleic acid sequence configured to bind to a target site of the single-stranded target sequence;
Y comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:150-155, or a complement of any of these;
L is a bond or comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:156-161, or a complement of any of these;
Z comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:162-178, or a complement of any of these; and
the nuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:100, 101, and 110.
18. A method of conducting a cleavage assay, the method comprising:
incubating a synthetic guiding component with a nuclease and a single-stranded target sequence, the synthetic guiding component including a targeting portion configured to bind and/or cleave the single-stranded target sequence; and
cleaving the single-stranded target sequence without a short DNA oligomer containing a proto-spacer adjacent motif (PAM) sequence (PAMmer);
wherein the synthetic guiding component comprises a structure having the formula (I):
W—X—Y-L-Z or a salt thereof, wherein:
W is an optional third portion comprising a nucleic acid sequence of from about 1 to 20 nucleic acids;
X is the targeting portion comprising a nucleic acid sequence configured to bind to a target site of the single-stranded target sequence;
Y is a first portion comprising a nucleic acid sequence configured to interact with a nuclease configured to cleave the single-stranded target sequence;
L is a linker; and
Z is a second portion comprising a nucleic acid sequence configured to interact with the nuclease and the first portion;
wherein the nuclease is a SauCas9 or CjeCas9 protein and the single-stranded target sequence is a single-stranded ribonucleic acid sequence.
19. The method of claim 18 , wherein the nuclease is a CjeCas9 protein.
20. The method of claim 18 , wherein the nuclease is a SauCas9 protein.
21. The method of claim 18 , wherein:
Y comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:150-155, 179-186, or a complement of any of these; and
Z comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:162-178, 192-212 or a complement of any of these.
22. The method of claim 18 , wherein:
L comprises a bond or a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:156-161, 187-191, 226-231, and 265-269, or a complement of any of these.
23. The method of claim 18 , wherein W and/or Y comprises one or more modified nucleic acids or bulges.
24. The method of claim 18 , wherein the single stranded target sequence recited for X is a single-stranded human mRNA target sequence or a single-stranded pathogen target sequence.
25. The method of claim 18 , X has a length of from about 15 to about 30 nucleotides.
26. The method of claim 18 wherein the nuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:100, 101, 104, 110, and 111.
27. The method of claim 26 , wherein the nuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 104, and 111.
28. The method of claim 18 , wherein W has a length of from 0 to about 20 nucleotides, X has a length of from about 10 to about 30 nucleotides, Y has a length of from about 10 to about 40 nucleotides, L has a length of from 0 to about 10 nucleotides, and Z has a length of from about 10 to about 90 nucleotides.
29. The method of claim 18 , wherein the synthetic guiding component has a length of from about 100 to about 200 nucleotides.
30. The method of claim 18 , further comprising detecting a label on the synthetic guiding component or a reporter on a nucleic acid substrate, wherein a detectable signal is provided upon recognition of the single-stranded target sequence by the synthetic guiding component.
31. The method of claim 18 , wherein the single-stranded ribonucleic acid sequence is the RNA sequence of a virus, the virus having a lifecycle consisting of solely RNA molecules.
32. The method of claim 18 , wherein the nuclease comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 100, 101, 104, 110, and 111.
33. The method of claim 18 , wherein Y comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:150-155, 179-186, or a complement of any of these; and
Z comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:162-178, 192-212 or a complement of any of these.