Methods Of Depleting Target Sequences Using CRISPR

• US 20160053304A1
• Filed: 07/17/2015
• Published: 02/25/2016
• Est. Priority Date: 07/18/2014
• Status: Abandoned Application

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Wurtzel, Omri, LoCascio, Samuel, Reddien, Peter
• Application Number:
  US14/802,886
• Publication Number:
  US 20160053304A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions

Abstract

Methods of depleting one or more target nucleic acid sequences using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) proteins (CRISPR/Cas) system are disclosed. Kits and methods of producing a library comprising select mRNA sequences using the CRISPR/Cas system are also disclosed.

Claims

1. A method of depleting one or more target nucleic acid sequences in a sample comprising the one or more target nucleic acid sequences and one or more non-target nucleic acid sequences, wherein each of the target nucleic acid sequences and the non-target nucleic acid sequences comprise a 5′

adapter and a 3′

adapter comprising;

(a) contacting the sample with;

i) one or more ribonucleic acid (RNA) sequences wherein all or a portion of each RNA sequence is complementary to all or a portion of at least one target nucleic acid sequence in the sample;

ii) a CRISPR associated (Cas) protein having nuclease activity; and

iii) a nucleic acid sequence that interacts with the Cas protein;

thereby producing a combination; and

(b) maintaining the combination under conditions in which the RNA sequences are allowed to hybridize to all or a portion of the target nucleic acid sequence to which each RNA sequence forms a complement thereby forming one or more base paired structures, and the one or more base-paired structures and the nucleic acid sequence that interacts with the Cas protein direct the Cas protein to deplete each of the target nucleic acid sequences;

thereby depleting the target nucleic acid in the sample.

2. The method of claim 1, further comprising isolating the one or more non-target nucleic acid sequences from the sample.

3. The method of claim 1, further comprising amplifying the non-target nucleic acid sequences in the sample.

5. The method of claim 1, wherein the Cas protein is Cas9.

6. The method of claim 1, wherein the RNA sequence is from about 10 base pairs to about 200 base pairs in length.

9. The method of claim 1, wherein the non-target nucleic acid in the sample is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

12. The method of claim 1, wherein the sample is a library, a cell lysate, or a biological sample.

16. The method of claim 1, wherein the one or more target nucleic acid sequences comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination thereof.

19. The method of claim 1, wherein the sample is contacted with the one or more RNA sequences, the Cas protein, and the nucleic acid sequence that interacts with Cas protein simultaneously or sequentially.

4. (canceled)

7. (canceled)

8. (canceled)

10. (canceled)

11. (canceled)

13. -15. (canceled)

17. (canceled)

18. (canceled)

20. (canceled)

21. A method of producing a mRNA library comprising:

(a) contacting a sample, wherein the sample comprises select mRNA to be included in the library and target nucleic acid sequences to be depleted from the library, and the select mRNA and target nucleic acid sequences each comprise a 5′

adapter and a 3′

adapter, with;

i) one or more ribonucleic acid (RNA) sequences wherein all or a portion of each RNA sequence is complementary to all or a portion of at least one target nucleic acid sequence in the sample;

ii) a CRISPR associated (Cas) protein having nuclease activity; and

iii) a nucleic acid sequence that interacts with the Cas protein;

thereby producing a combination;

(b) maintaining the combination under conditions in which the RNA sequences are allowed to hybridize to all or the portion of the target nucleic acid sequence to which each RNA sequence forms a complement thereby forming one or more base paired structures, and the one or more base paired structures and the nucleic acid sequence that interacts with the Cas protein direct the Cas protein to deplete each of the target nucleic acid sequences;

thereby producing a mRNA library comprising the select mRNA.

22. The method of claim 21, further comprising isolating the one or more non-target nucleic acid sequences from the sample.

23. The method of claim 21, further comprising amplifying the select mRNA in the sample.

25. The method of claim 21, wherein the Cas protein is Cas9.

26. The method of claim 21, wherein the RNA sequence is from about 10 base pairs to about 200 base pairs in length.

28. The method of claim 21, wherein the one or more target nucleic acid sequences comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or combinations thereof.

24. (canceled)

27. (canceled)

29. -32. (canceled)

33. A kit for producing a library of one or more non-target nucleic acid sequences from a sample comprising:

one or more ribonucleic acid (RNA) sequences wherein all or a portion of each RNA sequence is complementary to all or a portion of at least one or more target nucleic acid sequence in the sample that is to be excluded from the library;

a CRISPR associated (Cas) protein having nuclease activity;

a nucleic acid sequence that interacts with the Cas protein; and

one or more 5′

adapters and one or more 3′

adapters that bind to each of the one or more target nucleic acid sequences and each of the one or more non-target nucleic acid sequences in the sample.

34. The kit of claim 33, wherein the RNA sequence and the nucleic acid sequence that interacts with Cas protein are on the same sequence.

35. The kit of claim 33, wherein the RNA sequence is from about 10 base pairs to about 200 base pairs in length.

36. The kit of claim 33, wherein the sample comprises a virus.

37. The kit of claim 33, wherein the sample comprises one or more cells from an organism.

41. The kit of claim 33, wherein the Cas protein is Cas9.

42. The kit of claim 33, further comprising one or more components for an amplification reaction.

43. The kit of claim 33, wherein the library is a mRNA library.

38. -40. (canceled)