Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs
Provided are compositions and methods for detecting a target DNA (double stranded or single stranded) in a sample. In some embodiments, a subject method includes: (a) contacting the sample with: (i) a type V CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e); (ii) a guide RNA (comprising a region that binds to the type V CRISPR/Cas effector protein, and a guide sequence that hybridizes with the target DNA); and (iii) a detector DNA that is single stranded (i.e., a “single stranded detector DNA”) and does not hybridize with the guide sequence of the guide RNA; and (b) measuring a detectable signal produced by cleavage (by the type V CRISPR/Cas effector protein) of the single stranded detector DNA. Also provided are compositions and methods for cleaving single stranded DNAs (e.g., non-target ssDNAs), e.g., inside of a cell.
1. A method of multiplexed detection of target nucleic acids in a sample, the method comprising:
a) contacting the sample to:
i) a Cas12 protein;
ii) a first guide nucleic acid that hybridizes to a segment of a first target nucleic acid;
iii) a second guide nucleic acid that hybridizes to a segment of a second target nucleic acid; and
iv) a detector DNA molecule; and
b) assaying for a signal produced by cleavage of the detector DNA molecule by the Cas12 protein.
2. The method of claim 1 , further comprising contacting the sample to one or more additional guide nucleic acids that hybridize to a segment of one or more additional target nucleic acids.
3. The method of claim 1 , wherein the first target nucleic acid, the second target nucleic acid, or both, are single stranded.
4. The method of claim 1 , wherein the first target nucleic acid, the second target nucleic acid, or both, are double stranded.
5. The method of claim 1 , wherein the first target nucleic acid, the second target nucleic acid, or both, is present in the sample at a concentration as low as 200 fM.
6. The method of claim 1 , wherein the first target nucleic acid, the second target nucleic acid, or both, is present in the sample at one or more copies per 10 7 non-target nucleic acids.
7. The method of claim 1 , wherein the Cas12 protein exhibits trans cleavage activity subsequent to binding to the first target nucleic acid or the second target nucleic acid.
8. The method of claim 1 , wherein the first target nucleic acid, the second target nucleic acid, or both, are associated with a disease state.
9. The method of claim 1 , wherein the first target nucleic acid, the second target nucleic acid, or both, are viral nucleic acids or bacterial nucleic acids.
10. The method of claim 9 , wherein the viral nucleic acids are papovavirus, human papillomavirus (HPV), hepadnavirus, Hepatitis B Virus (HBV), herpesvirus, varicella zoster virus (VZV), Epstein Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus, adenovirus, poxvirus, parvovirus, influenza virus, or respiratory syncytial virus nucleic acid.
11. The method of claim 1 , wherein the first target nucleic acid is a nucleic acid from a first viral strain or a first bacterial strain and the second target nucleic acid is a nucleic acid from a second viral strain or a second bacterial strain.
12. The method of claim 11 , wherein the first viral strain is a first strain of influenza virus and the second viral strain is a second strain of influenza virus.
13. The method of claim 11 , wherein the first viral strain is a first strain of influenza virus and the second viral strain is a strain of respiratory syncytial virus.
14. The method of claim 1 , wherein the first target nucleic acid is a nucleic acid from a first viral strain or a first bacterial strain and the second target nucleic acid is a nucleic acid from the first viral strain or the first bacterial strain.
15. The method of claim 1 , wherein the second guide nucleic acid is a positive control guide nucleic acid and the second target nucleic acid is a positive control nucleic acid.
16. The method of claim 15 , wherein the first target nucleic acid and the second target nucleic acid are different.
17. The method of claim 1 , wherein the first target nucleic acid, the second target nucleic acid, or both, comprise a single nucleotide polymorphism.
18. The method of claim 1 , wherein the first target nucleic acid, the second target nucleic acid, or both, are from a human cell.
19. The method of claim 1 , wherein the first target nucleic acid, the second target nucleic acid, or both are human fetal nucleic acids, cancer cell nucleic acids, or a combination thereof.
20. The method of claim 1 , wherein the Cas12 protein is a Cas12a polypeptide, a Cas12b polypeptide, a Cas12c polypeptide, a Cas12d polypeptide, or a Cas12e polypeptide.
21. The method of claim 1 , wherein the sample comprises nucleic acids derived from a cell or a cell lysate.
22. The method of claim 21 , further comprising contacting the cell lysate, or a portion thereof, with amplification reagents.
23. The method of claim 1 , wherein the sample comprises nucleic acids derived from blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage, sputum, a fecal sample, cerebrospinal fluid, a fine needle aspirate, a buccal swab, a cervical swab, a nasal swab, interstitial fluid, synovial fluid, nasal discharge, tears, buffy coat, or a mucous membrane sample.
24. The method of claim 1 , further comprising determining an amount of the first target nucleic acid and an amount of the second target nucleic acid present in the sample.
25. The method of claim 1 , wherein the assaying for the signal produced by cleavage of the detector DNA molecule comprises one or more of: visual based detection, sensor-based detection, color detection, gold nanoparticle based detection, fluorescence polarization, colloid phase transition, colloid phase dispersion, electrochemical detection, and semiconductor-based sensing.
26. The method of claim 1 , wherein the signal is a reduction in signal intensity after cleavage of the detector DNA molecule.
27. The method of claim 1 , wherein the signal is an increase in signal intensity after cleavage of the detector DNA molecule.
28. The method of claim 1 , wherein the detector DNA molecule comprises one or more fluorescence-emitting dye pairs.
29. The method of claim 28 , wherein the one or more fluorescence-emitting dye pairs includes a fluorescence resonance energy transfer (FRET) pair, a quencher and fluorophore pair, or a combination thereof.
30. The method of claim 29 , wherein cleavage of the detector DNA molecule separates the fluorescence resonance energy transfer (FRET) pair from one another to produce the signal.
31. The method of claim 29 , wherein cleavage of the detector DNA molecule separates the quencher and fluorophore pair from one another to produce the signal.
32. The method of claim 1 , wherein the first guide nucleic acid, the second guide nucleic acid, or both comprise a constant region comprising the nucleotide sequence of SEQ ID NO:11.
33. The method of claim 1 , wherein the first target nucleic acid, the second target nucleic acid, or both comprise amplified nucleic acids.
34. The method of claim 33 , wherein the amplified nucleic acids are generated by polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), quantitative PCR (qPCR), reverse transcription qPCR (RT-qPCR), nested PCR, multiplex PCR, asymmetric PCR, touchdown PCR, random primer PCR, hemi-nested PCR, polymerase cycling assembly (PCA), colony PCR, ligase chain reaction (LCR), digital PCR, methylation specific-PCR (MSP), co-amplification at lower denaturation temperature-PCR (COLD-PCR), allele-specific PCR, intersequence-specific PCR (ISS-PCR), whole genome amplification (WGA), inverse PCR, thermal asymmetric interlaced PCR (TAIL-PCR), loop-mediated amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), or isothermal multiple displacement amplification (IMDA).
35. The method of claim 1 , wherein the first target nucleic acid, the second target nucleic acid, or both are RNA.
36. The method of claim 1 , wherein the first guide nucleic acid and the second guide nucleic acid are comprised of RNA and the detector DNA molecule is comprised of single stranded DNA.
37. The method of claim 36 , wherein the first guide nucleic acid or the second guide nucleic acid comprise one or more modifications.
38. The method of claim 37 , wherein the one or more modifications comprise a base modification, a base substitution, a polynucleotide mimetic, a backbone modification, a non-natural internucleoside linkage, a sugar modification, or a sugar substitution.
39. The method of claim 1 , wherein the segment of the first target nucleic acid is between 16-24 base pairs and the segment of the second target nucleic acid is between 16-24 base pairs.
40. The method of claim 1 , wherein the first target nucleic acid is a nucleic acid from a first viral variant or a first bacterial variant and the second target nucleic acid is a nucleic acid from a second viral variant or a second bacterial variant.
41. The method of claim 1 , wherein the first target nucleic acid is a nucleic acid from a first viral variant or a first bacterial variant and the second target nucleic acid is a nucleic acid from the first viral variant or the first bacterial variant.