NUCLEIC ACID FLUORESCENCE DETECTION

Abstract

The invention relates to a nucleic acid detection system, a diagnostic device, use of the nucleic acid detection system as a diagnostic agent, a kit-of-parts for detecting nucleic acids, a method for detecting nucleic acids, and a method for diagnosing a disease state of a subject. The nucleic acid detection system comprises a CRISPR-Cas system which comprises an effector protein and one or more guide RNAs having a guide sequence, the guide sequence being capable of targeting the effector protein to a target sequence of a target, and the effector protein exhibiting target-activated nucleic acid cleavage activity capable of cleaving nucleic acid reporter molecules to generate nucleic acid fragments; and a polymerase exhibiting catalytic activity capable of transferring nucleotides to the fragments to form polynucleotide tails, wherein preferably the detection system is a nucleic acid fluorescence detection system.

Claims

1. A nucleic acid detection system, comprising: a CRISPR-Cas system which comprises an effector protein and one or more guide RNAs having a guide sequence, the guide sequence being capable of targeting the effector protein to a target sequence of a target nucleic acid, and the effector protein exhibiting target-activated nucleic acid cleavage activity capable of cleaving nucleic acid reporter molecules to generate nucleic acid fragments; and a polymerase exhibiting catalytic activity capable of transferring nucleotides to the nucleic acid fragments to form polynucleotide tails attached to the nucleic acid fragments.

2. The nucleic acid detection system of claim 1, wherein the polynucleotide tails comprise binding sites capable of hybridising with polynucleotides.

3. The nucleic acid detection system of claim 1, wherein the detection system is a nucleic acid fluorescence detection system.

4. The nucleic acid detection system of claim 1, wherein the CRISPR-Cas system is a class 2 CRISPR-Cas system.

5. The nucleic acid detection system of claim 1, wherein the polymerase comprises DNA polymerase and/or RNA polymerase.

6. The nucleic acid detection system of claim 1, further comprising nucleotides and/or derivatives thereof.

7. The nucleic acid detection system of claim 6, wherein the nucleotides comprise deoxythymidine triphosphate.

8. The nucleic acid detection system of claim 1, wherein the polynucleotide tails comprise metal-binding sites capable of chelating metal.

9. The nucleic acid detection system of claim 8, wherein the metal is copper.

10. (canceled)

11. The nucleic acid detection system of claim 1, wherein the target nucleic acids is a microbial nucleic acids.

12. The nucleic acid detection system of claim 1, further comprising nucleic acid reporter molecules.

13. (canceled)

14. A diagnostic device, comprising one or more nucleic acid detection systems of claim 1, and optionally comprising a source of electromagnetic radiation.

15. A kit-of-parts for detecting nucleic acids, the kit-of-parts comprising: i) a first container (A) which comprises: a CRISPR-Cas system, ii) a second container (B) which comprises: a detectable compound, ii) a third container (C) which comprises: a reductant, and iv) optionally a fourth container (D) which comprises: a source of electromagnetic radiation.

16. The kit-of-parts of claim 15, wherein the detectable compound comprises a metal.

17. The kit-of-parts of claim 15, wherein the detectable compound comprises a polynucleotide.

18. A method for detecting a target nucleic acid using the CRISPR-Cas system as defined in claim 1, nucleic acid reporter molecules, nucleotides, and detectable compounds; the method comprising: i) targeting the effector protein with the guide sequence to the target sequence of the target nucleic acid, thereby target activating the nucleic acid cleavage activity of the effector protein; ii) cleaving the nucleic acid reporter molecules with the activated effector protein to generate nucleic acid fragments; iii) adding the polymerase and the nucleotides form a polynucleotide tail attached to the nucleic acid fragments; and iv) binding the detectable compounds to the polynucleotide tail, thereby forming a detectable cluster.

19. The method of claim 18, wherein the detectable compound comprises a metal that binds to the polynucleotide tail, and the detectable cluster is a detectable metal cluster, and wherein the method optionally further comprises: v) exposing the detectable metal cluster to electromagnetic radiation.

20. The method of claim 18, wherein the detectable compound comprises a labelled polynucleotide that binds to the polynucleotide tail, and the detectable cluster is a detectable hybrid cluster.

21. The method of claim 18, wherein the method is a method for detecting and/or monitoring the target nucleic acid in a subject.

22. The method of claim 18, wherein the method is a method for diagnosing a disease state of a subject.

23. The method of claim 18, wherein the target nucleic acids are is obtained from a biological sample or an environmental sample.

24. The method of claim 18, wherein presence of the target nucleic acid diagnostic for a disease state.

Description

EXAMPLE 1

Feasibility Study

[0156] A strategy for specific Cas12a-dependent poly(thymine) formation is given in FIG. 1. The strategy is divided in essentially three steps. To achieve Cas12a trans-cleavage activation, Lachnospiraceae bacterium Cas12a (NEB), a plasmid containing a gene encoding part of Anthrax Lethal Factor (ALF; aa 253-380) and the three corresponding crRNAs were employed (see Materials). In order to combine the three-step process in a one tube system, a single reaction buffer needed to be established. All three key components, i.e., Cas12a, TdT and copper nanoclusters function optimally in weakly basic conditions. TdT is inhibited by high concentrations of chloride ions present in recommended Cas12a buffers, therefore the reaction was performed in an acetate containing buffer (potassium-acetate, tris-acetate and magnesium acetate pH 7.9, see Materials). As illustrated in FIG. 2 a very strong fluorescent signal is emitted in the presence of the complete reaction mix. Each of the three crRNAs caused Cas12a activation and a strong fluorescent signal. Leaving out any of the components in the reaction, crRNA, ALF gene, Cas12a enzyme or the blocked reporter, did not result in an observable signal. Thus, both enzymes Cas12a and TdT perform well in these buffer conditions. The presence of 0.25 mM CoCl.sub.2, which is a necessary cofactor for TdT added in step 2, is compatible with downstream copper nanocluster formation.

SENSITIVITY STUDY

[0157] Having established that it is possible to perform the three-step process in a single tube, the sensitivity of the detection method was tested using serially diluted plasmid DNA (FIG. 3). Based on both the fluorescence measurement and visual assessment, the detection limit of the system as described herein was evaluated at 10 picomolar. To investigate the reaction time, identical reaction mixtures were incubated over varying time intervals. Cas12a was added to each of the tubes and left for time intervals of 15, 30 and 60 min. Subsequently TdT was added and incubated for time intervals of 1, 2 and 3 hours. The result, given in FIG. 4, shows that for both enzymes maximal incubation times (1 hr for Cas12a, and 3 hours for TdT) promote the signal intensity. The shortest times allowing detection above background using the fluorescence plate reader is 15 min for Cas12a and 1 hr for TdT, but the signal is hard to discriminate by eye.