CRISPR effector system based diagnostics

• US 10,266,887 B2
• Filed: 03/09/2018
• Issued: 04/23/2019
• Est. Priority Date: 12/09/2016
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Abudayyeh, Omar, Collins, James Joseph, Gootenberg, Jonathan, Zhang, Feng, Lander, Eric S.
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US15/917,549
• Publication Number:
  US 20180340219A1
• Time in Patent Office: 410 Days
• Field of Search: None
• CPC Class Codes: C12N 15/113 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 Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: G01N 33/00 Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

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]X₁X₂X₃H sequence, wherein X₁ is R, S, D, E, Q, N, G, or Y, and X₂ is independently I, S, T, V, or L, and X₃ 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.