CRISPR effector system based diagnostics
The embodiments disclosed herein utilized RNA targeting effectors to provide a robust CRISPR-based diagnostic with attomolar sensitivity. Embodiments disclosed herein can detect broth DNA and RNA with comparable levels of sensitivity and can differentiate targets from non-targets based on single base pair differences. Moreover, the embodiments disclosed herein can be prepared in freeze-dried format for convenient distribution and point-of-care (POC) applications. Such embodiments are useful in multiple scenarios in human health including, for example, viral detection, bacterial strain typing, sensitive genotyping, and detection of disease-associated cell free DNA.
1. A method for detecting target nucleic acids in samples comprising:
contacting one or more samples with
reagents for amplifying one or more target sequences;
a Cas13;
at least one guide polynucleotide comprising a guide sequence capable of binding the target sequences, and designed to form a complex with the Cas13; and
an RNA-based masking construct comprising a non-target sequence, wherein the Cas13 exhibits collateral RNase activity and cleaves the non-target sequence of the RNA-based masking construct once activated by the target sequences; and detecting a signal from cleavage of the non-target sequence, thereby detecting the one or more target sequences in the sample.
2. The method of claim 1 , wherein the one or more target sequences is a target DNA and the method further comprises contacting the target DNA with a primer comprising an RNA polymerase site and an RNA polymerase.
3. The method of claim 2 , wherein the reagents for amplifying the one or more target sequences comprise isothermal amplification reaction reagents.
4. The method of claim 3 , wherein the isothermal amplification reagents comprise nucleic-acid sequence-based amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification, or nicking enzyme amplification reagents.
5. The method of claim 1 , wherein the Cas13 comprises one or more higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains.
6. The method of claim 5 , wherein the one or more HEPN domains comprise a RxxxxH motif sequence.
7. The method of claim 6 , wherein the RxxxxH motif comprises a R[N/H/K]X 1 X 2 X 3 H sequence, wherein X 1 is R, S, D, E, Q, N, G, or Y, and X 2 is independently I, S, T, V, or L, and X 3 is independently L, F, N, Y, V, I, S, D, E, or A.
8. The method of claim 1 , wherein the Cas13 is a Cas13a, Cas13b, Cas13c, or combination thereof.
9. The method of claim 1 , wherein the masking construct suppresses generation of a detectable positive signal until cleaved or deactivated, or masks a detectable positive signal, or generates a detectable negative signal until the masking construct is deactivated or cleaved.
10. The method of claim 9 , wherein the masking construct comprises:
a. a silencing RNA that suppresses generation of a gene product encoded by a reporting construct, wherein the gene product generates the detectable positive signal when expressed;
b. a ribozyme that generates the negative detectable signal, and wherein the positive detectable signal is generated when the ribozyme is deactivated;
c. a ribozyme that converts a substrate to a first color and wherein the substrate converts to a second color when the ribozyme is deactivated;
d. an aptamer and/or comprises a polynucleotide-tethered inhibitor;
e. a polynucleotide to which a detectable ligand and a masking component are attached;
f. a nanoparticle held in aggregate in a solution by bridge molecules, wherein at least a portion of the bridge molecules comprises a polynucleotide, and wherein the solution undergoes a color shift when the nanoparticle is disbursed in solution;
g. a quantum dot or fluorophore linked to one or more quencher molecules by a linking molecule, wherein at least a portion of the linking molecule comprises a polynucleotide;
h. a polynucleotide in complex with an intercalating agent, wherein the intercalating agent changes absorbance upon cleavage of the polynucleotide; or
i. two fluorophores tethered by a polynucleotide that undergo a shift in fluorescence when released from the polynucleotide.
11. The method of claim 10 , wherein the aptamer
a. comprises a polynucleotide-tethered inhibitor that sequesters an enzyme, wherein the enzyme generates a detectable signal upon release from the aptamer or polynucleotide-tethered inhibitor by acting upon a substrate;
b. is an inhibitory aptamer that inhibits an enzyme and prevents the enzyme from catalyzing generation of a detectable signal from a substrate or wherein the polynucleotide-tethered inhibitor inhibits an enzyme and prevents the enzyme from catalyzing generation of a detectable signal from a substrate; or
c. sequesters a pair of agents that when released from the aptamer combine to generate a detectable signal.
12. The method of claim 10 , wherein the nanoparticle is a colloidal metal.
13. The method of claim 1 , wherein the guide sequence comprises a mismatch to the one or more target sequences.
14. The method of claim 13 , wherein the mismatch is up- or downstream of a single nucleotide variation in the guide sequence.