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 .- 45 . (canceled)
46 . A method comprising:
a) contacting a DNA editing enzyme to a reporter, wherein the DNA editing enzyme comprises a guide nucleic acid having a DNA recognition sequence, wherein the DNA editing enzyme comprises DNA endonuclease enzyme activity, and wherein the reporter does not comprise the DNA recognition sequence; and
b) assaying for an output of the reporter.
47 . The method of claim 46 , wherein the DNA editing enzyme exhibits double stranded DNA cleavage activity.
48 . The method of claim 46 , wherein the DNA editing enzyme exhibits single stranded DNA cleavage activity.
49 . The method of claim 46 , wherein the DNA editing enzyme exhibits trans cleavage activity subsequent to binding to a nucleic acid encoding a sequence that is reverse complementary to the DNA recognition sequence.
50 . The method of claim 46 , wherein the DNA recognition sequence is reverse complementary to a nucleic acid associated with a genetic condition.
51 . The method of claim 50 , wherein the genetic condition is cancer.
52 . The method of claim 46 , wherein the DNA recognition sequence is reverse complementary to a nucleic acid from a virus, bacterium, or parasite.
53 . The method of claim 46 performed on a paper substrate.
54 . The method of claim 46 , wherein the DNA editing enzyme is a programmable nuclease.
55 . The method of claim 46 , wherein the DNA editing enzyme is a type V CRISPR/Cas effector protein.
56 . The method of claim 46 , wherein the DNA editing enzyme comprises a RuvC nuclease domain
57 . The method of claim 46 , wherein the DNA editing enzyme is a Cas12 family effector protein.
58 . The method of claim 46 , wherein the DNA editing enzyme is selected from a Cas12a polypeptide, a Cas12b polypeptide, a Cas12c polypeptide, a Cas12d polypeptide, a Cas12e polypeptide, a C2c4 polypeptide, a C2c8 polypeptide, a C2c5 polypeptide, a C2c10 polypeptide, and a C2c9 polypeptide.
59 . The method of claim 46 , wherein the reporter is a nucleic acid comprising a detection moiety.
60 . The method of claim 46 , wherein the DNA recognition sequence correlates to a diseased state.
61 . The method of claim 46 , wherein the DNA recognition sequence correlates to a non-diseased state.
62 . The method of claim 46 , comprising contacting the DNA editing enzyme to a target DNA prior to assaying for the output of the reporter, wherein the target DNA encodes a sequence that is reverse complementary to the DNA recognition sequence.
63 . The method of claim 61 , wherein the target DNA is single stranded DNA.
64 . The method of claim 61 , wherein the target DNA is double stranded DNA.
65 . The method of claim 61 , further comprising amplifying the target DNA.
66 . The method of claim 64 , wherein the amplifying comprises thermal cycling amplification.
67 . The method of claim 64 , wherein the amplifying is isothermal amplifying.
68 . The method of claim 64 , wherein the amplifying is by isothermal recombinase polymerase amplification (RPA), transcription mediated amplification (TMA), strand displacement amplification (SDA), helicase dependent amplification (HDA), loop mediated amplification (LAMP), rolling circle amplification (RCA), single primer isothermal amplification (SPIA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), or improved multiple displacement amplification (IMDA).
69 . The method of claim 61 further comprising determining an amount of the target DNA in the sample.