Molecular sensing platform and methods of use

1 . A method of target-nucleic acid-specific generation of a crRNA-encoding nucleic acid in a sample putatively containing the target nucleic acid, the method comprising:

a. providing the sample putatively containing the target nucleic acid;

b. contacting the sample with a system, the system comprising:

i) a primer pair comprising a promoter primer and a crRNA primer,

the promoter primer comprising, from 5′ to 3′, a transcriptional promoter, and a proximal detection target segment that has, or is complementary to, the sequence of a proximal portion of the target nucleic acid, and

the crRNA primer comprising, from 5′ to 3′, a crRNA encoding segment that is a sequence encoding a crRNA or the reverse complement of a sequence encoding a crRNA, and a distal detection target segment that has, or is complementary to, the sequence of a distal portion of the target nucleic acid,

wherein the target segments in each primer permit amplification from the detection target nucleic acid;

ii) a polymerase; and

iii) components for nucleic acid amplification; and

c. incubating the sample and the system of step b. under conditions for target-specific amplification of the target sequence to generate a crRNA-encoding nucleic acid.

2 . The method of claim 1 for detecting a target nucleic acid in a sample, the method further comprising:

d. optionally, separating the crRNA-encoding nucleic acid, optionally wherein separating the crRNA-encoding nucleic acid comprises i) isolating the crRNA-encoding nucleic acid, from the system; or ii) inactivating the primers;

e. contacting the crRNA-encoding nucleic acid with an RNA polymerase and components for transcription;

f. incubating the crRNA-encoding nucleic acid, RNA polymerase and components for transcription under conditions for the generation of a crRNA;

g. contacting the crRNA with a CRISPR-Cas protein, optionally the CRISPR-Cas protein Cas12a;

h. incubating the crRNA and CRISPR-Cas protein under conditions to allow the binding of the crRNA to the CRISPR-Cas protein to generate an active CRISPR-Cas effector protein;

i. contacting the active CRISPR-Cas effector protein with a signal-generating CRISPR-sensitive reporter;

j. incubating the CRISPR-Cas effector protein and signal-generating CRISPR-sensitive reporter under conditions to allow the generation of signal from the signal-generating CRISPR-sensitive reporter; and

k. detecting the presence or absence of signal.

3 . The method of claim 1 , wherein the sample is a) a biological sample, or b) an environmental sample, c) a sample comprising a barcode, or a combination thereof, and/or wherein the target nucleic acid is purified or amplified from the sample prior to the application of the method.

4 . The method of claim 3 wherein the biological sample is obtained from a tissue sample, saliva, blood, plasma, sera, stool, urine, semen, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, skin swab, or mucosal membrane surface, or the environmental sample is or is obtained from a food sample, a beverage sample, a surface, a soil sample, a water sample, exposure to atmospheric air or other gas sample.

5 . The method of claim 1 , wherein the polymerase is a DNA polymerase, optionally selected from Bsu, IsoPol, AMV-RT or Q5, or any DNA polymerase, suitable for use in isothermal amplification, optionally selected from HDA (Helicase-dependent amplification), LAMP (Loop-mediated Isothermal Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), RPA (Recombinase Polymerase Amplification), NEAR (Nicking Enzyme Amplification Reaction), or suitable for PCR or combinations thereof.

6 . The method of claim 1 , wherein target nucleic acid is an RNA and the polymerase is a reverse transcriptase optionally AMV-RT.

7 . The method of claim 2 , wherein

a. the promoter primer comprises a T7 promoter and the RNA polymerase is T7 polymerase;

b. the promoter primer comprises a T3 promoter and the RNA polymerase is T3 polymerase; or

c. the promoter primer comprises a SP6 promoter and the RNA polymerase is SP6 polymerase.

8 . The method of claim 2 , wherein the signal-generating CRISPR-sensitive reporter is a molecular beacon (MB), optionally wherein the molecular beacon which comprises a CRISPR sensitive nucleic acid linker, a fluorophore and a quencher, wherein the CRISPR sensitive nucleic acid linker is double stranded and optionally wherein the fluorophore and the quencher are opposite.

9 . The method of claim 2 , wherein the signal-generating CRISPR-sensitive reporter is a CRISPR-sensitive DNA sensor, and the method further comprises in step i. contacting the active CRISPR-Cas effector protein with components for function-restoring repair of the signal-generating reporter and incubating the active CRISPR-Cas effector protein, signal-generating CRISPR-sensitive reporter, and components under conditions to allow a function restoring repair of the signal generating reporter.

10 . The method of claim 9 , wherein the CRISPR-sensitive DNA sensor comprises:

a. a non-functional CRISPR-sensitive DNA reporter construct comprising a non-functional expression cassette with at least one CRISPR target site inserted or naturally present in the expression cassette, the non-functional expression cassette having a reporter construct upstream end upstream of the CRISPR target site and a reporter construct downstream end downstream of the CRISPR target site, and

b. at least one function-restoring nucleic acid, the function-restoring nucleic acid comprising a downstream flanking end and a function restoring repair insert, optionally an upstream flanking end, optionally comprising a promoter,

wherein the upstream flanking end interfaces with the reporter construct upstream end and/or the downstream flanking end interfaces with the reporter construct downstream end and one or both of the flanking ends permitting insertion or ligation of the function restoring repair insert into/to the reporter construct when the CRISPR target site is actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal, or

the CRISPR-sensitive DNA sensor comprises:

a. a non-functional DNA reporter construct comprising a non-functional expression cassette, the non-functional expression cassette having a single stranded part;

b. at least one function-restoring nucleic acid optionally supplemented dsDNA, the function-restoring nucleic acid comprising:

i. a CRISPR target site inserted or naturally present in the function restoring nucleic acid, and

ii. a function restoring repair insert complementary to the single stranded part of the non-functional DNA reporter construct, the function restoring insert being releasable upon CRISPR mediated cleavage of the function restoring nucleic acid;

wherein the function restoring repair insert interfaces (hybridizes) with the reporter construct single stranded part permitting insertion or ligation of the function restoring repair insert into/to the reporter construct when the CRISPR target site is actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal; or

the CRISPR-sensitive DNA sensor comprises a non-functional CRISPR-sensitive DNA reporter construct, the reporter construct comprising:

a. a promoter,

b. a reporter cassette,

c. a function-blocking region optionally in the promoter, or within a transcription start site or in a coding region of the reporter cassette,

d. CRISPR-Cas target sites that flank the function blocking region;

e. a reporter construct upstream end upstream of the function-blocking region; and

f. a reporter construct downstream end downstream of the function-blocking region;

wherein the upstream end is capable of interfacing with the downstream end to permit function-restoring repair of the reporter construct when the CRISPR target sites are actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal.

11 . The method of claim 2 ,

wherein the signal-generating CRISPR-sensitive reporter in step i. is a CRISPR-sensitive DNA sensor comprising:

i) a non-functional CRISPR-sensitive DNA reporter construct comprising a non-functional expression cassette with at least one CRISPR target site inserted or naturally present in the expression cassette, the non-functional expression cassette having a reporter construct upstream end upstream of the CRISPR target site and a reporter construct downstream end downstream of the CRISPR target site, and

ii) at least one function-restoring nucleic acid, the function-restoring nucleic acid comprising a downstream flanking end and a function restoring repair insert, optionally an upstream flanking end,

wherein the upstream flanking end interfaces with the reporter construct upstream end and/or the downstream flanking end interfaces with the reporter construct downstream end and one or both of the flanking ends permitting insertion or ligation of the function restoring repair insert into/to the reporter construct when the CRISPR target site is actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal;

and the method further comprises in step i incubating the CRISPR-Cas effector protein and signal-generating CRISPR-sensitive reporter under conditions to allow function restoring repair of the signal-generating CRISPR-sensitive reporter.