CRISPR effector system based diagnostics

Patent metadata

Field	Value
Publication number	US-10266887-B2
Application number	US-201815917549-A
Country	US
Kind code	B2
Filing date	Mar 9, 2018
Priority date	Dec 9, 2016
Publication date	Apr 23, 2019
Grant date	Apr 23, 2019

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College

Inventors

• Abudayyeh Omar
• Collins James Joseph
• Gootenberg Jonathan
• Zhang Feng
• Lander Eric S

Classifications

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6811

  Selection methods for production or design of target specific oligonucleotides or binding molecules · CPC title

• C12N2310/16

  Aptamers · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title