Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs
View Patent ↗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 detecting a target DNA in a sample, the method comprising:
(a) contacting the sample with:
(i) a type V CRISPR/Cas effector protein;
(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 labeled detector DNA that is single stranded and does not hybridize with the guide sequence of the guide RNA; and
(b) measuring a detectable signal produced by cleavage of the labeled detector DNA by the type V CRISPR/Cas effector protein, thereby detecting the target DNA.
2. The method of claim 1 , comprising contacting the sample with a precursor guide RNA array, wherein the type V CRISPR/Cas effector protein cleaves the precursor guide RNA array to produce said guide RNA.
3. The method of claim 1 , wherein the target DNA is single stranded.
4. The method of claim 1 , wherein the target DNA is double stranded.
5. The method of claim 1 , wherein the target DNA is viral DNA or bacterial DNA.
6. The method of claim 1 , wherein the target DNA is 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, or parvovirus DNA.
7. The method of claim 1 , wherein the target DNA is from a human cell.
8. The method of claim 1 , wherein the target DNA is human fetal or cancer cell DNA.
9. The method of claim 1 , wherein the type V CRISPR/Cas effector protein is a Cas12a (Cpf1) or Cas12b (C2c1) protein.
10. The method of claim 1 , wherein the sample comprises DNA from a cell lysate.
11. The method of claim 1 , wherein the sample comprises cells.
12. The method of claim 1 , wherein the sample is a blood, serum, plasma, urine, aspirate, or biopsy sample.
13. The method of claim 1 , comprising determining an amount of the target DNA present in the sample.
14. The method of claim 1 , wherein said measuring a detectable signal comprises one or more of: visual based detection, sensor based detection, color detection, gold nanoparticle based detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, and semiconductor-based sensing.
15. The method of claim 1 , wherein the labeled detector DNA comprises a modified nucleobase, a modified sugar moiety, and/or a modified nucleic acid linkage.
16. The method of claim 1 , further comprising detecting a positive control target DNA in a positive control sample, the detecting comprising:
(c) contacting the positive control sample with:
(i) the type V CRISPR/Cas effector protein;
(ii) a positive control guide RNA comprising: a region that binds to the type V CRISPR/Cas effector protein, and a positive control guide sequence that hybridizes with the positive control target DNA; and
(iii) a labeled detector DNA that is single stranded and does not hybridize with the positive control guide sequence of the positive control guide RNA; and
(d) measuring a detectable signal produced by cleavage of the labeled detector DNA by the type V CRISPR/Cas effector protein, thereby detecting the positive control target DNA.
17. The method of claim 1 , wherein the detectable signal is detectable in less than 45 minutes.
18. The method of claim 1 , wherein the detectable signal is detectable in less than 30 minutes.
19. The method of claim 1 , further comprising amplifying the target DNA in the sample by loop-mediated isothermal 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).
20. The method of claim 1 , wherein target DNA in the sample is present at a concentration of less than 10 aM.