Multi-effector CRISPR based diagnostic systems

Patent metadata

Field	Value
Publication number	US-11898142-B2
Application number	US-201816645571-A
Country	US
Kind code	B2
Filing date	Sep 7, 2018
Priority date	Sep 9, 2017
Publication date	Feb 13, 2024
Grant date	Feb 13, 2024

Abstract

The embodiments disclosed herein utilized RNA targeting effectors to provide a robust CRISPR-based diagnostic with attomolar sensitivity. Embodiments disclosed herein can detect both 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 nucleic acid detection system comprising:
a) a detection CRISPR system comprising:
i. a CRISPR effector protein having collateral cleavage activity,
ii. one or more guide RNAs designed to bind to corresponding target nucleic acids, and
iii. one or more signal amplification Type IIIa CRISPR effector proteins; and

b) one or more RNA-based masking constructs.

2. A polypeptide detection system comprising:
a) a detection CRISPR system comprising:
i. a CRISPR effector protein having collateral cleavage activity,
ii. one or more guide RNAs designed to bind to corresponding trigger RNAs, and
iii. one or more signal amplification Type IIIa CRISPR effector proteins;

b) one or more RNA-based masking constructs; and
c) one or more polypeptide detection aptamers designed to bind to corresponding target polypeptides and comprising a masked RNA polymerase promoter binding site or a masked primer binding site.

3. The system of claim 1 , wherein the system further comprises nucleic acid amplification reagents.

4. The system of claim 1 , wherein the signal amplification Type IIIa CRISPR protein is a Csm6 protein.

5. The system of claim 1 , wherein the one or more signal amplification Type IIIa CRISPR effector proteins comprises one or more of Csm6, Csx28, Csx27 or any combination thereof.

6. The system of claim 1 , wherein the target nucleic acid is a target DNA and the system further comprises a primer that binds the target DNA and comprises an RNA polymerase promoter.

7. The system of claim 1 , wherein the CRISPR effector protein having collateral cleavage activity is CRISPR RNA-targeting effector protein; wherein the CRISPR RNA-targeting effector protein optionally comprises one or more HEPN domains; wherein the one or more HEPN domains optionally comprise a RxxxxH motif sequence; wherein the RxxxxH motif sequence optionally comprises a R{N/H/K]X1X2X3H sequence; and 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 system of claim 7 , wherein the CRISPR RNA-targeting effector protein is C2c2 or Cas13b, and wherein the C2c2 is within 20 kb of a Cas 1 gene.

9. The system of claim 8 , wherein the C2c2 effector protein is from an organism of a genus selected from the group consisting of: Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, Campylobacter , and Lachnospira;
wherein the C2c2 effector protein is optionally from an organism selected from the group consisting of: Leptotrichia shahii; Leptotrichia wadei (Lw2); Listeria seeligeri; Lachnospiraceae bacterium MA2020 ; Lachnospiraceae bacterium NK4A179 ; [Clostridium] aminophilum DSM 10710 ; Carnobacterium gallinarum DSM 4847 ; Carnobacterium gallinarum DSM 4847 (second CRISPR Loci); Paludibacter propionicigenes WB4; Listeria weihenstephanensis FSL R9-0317 ; Listeriaceae bacterium FSL M6-0635 ; Leptotrichia wadei F0279; Rhodobacter capsulatus SB 1003; Rhodobacter capsulatus R121 ; Rhodobacter capsulatus DE442 ; Leptotrichia buccalis C-1013-b; Herbinix hemicellulosilytica; [Eubacterium] rectale; Eubacteriaceae bacterium CHKCI004 ; Blautia sp. Marseille-P2398 ; Leptotrichia sp. oral taxon 879 str. F0557 ; Lachnospiraceae bacterium NK4A144 ; Chloroflexus aggregans; Demequina aurantiaca; Thalassospira sp. TSL5-1 ; Pseudobutyrivibrio sp. OR37 ; Butyrivibrio sp. YAB3001 ; Blautia sp. Marseille-P2398 ; Leptotrichia sp. Marseille-P3007 ; Bacteroides ihuae; Porphyromonadaceae bacterium KH3CP3RA; Listeria riparia ; and Insolitispirillum peregrinum;
wherein the C2c2 effector protein is optionally a L. wadei F0279 or L. wadei F0279 (Lw2) C2c2 effector protein.

10. The system of claim 1 , wherein the one or more RNA-based masking constructs suppresses generation of a detectable positive signal; and
wherein the one or more RNA-based masking constructs optionally suppresses generation of a detectable positive signal by masking the detectable positive signal, or generating a detectable negative signal instead; or
wherein the one or more RNA-based masking constructs optionally comprises 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.

11. The system of claim 10 , wherein the one or more RNA-based masking constructs is a ribozyme that generates the negative detectable signal, and wherein the positive detectable signal is generated when the ribozyme is deactivated, optionally wherein the ribozyme converts a substrate to a first color and wherein the substrate converts to a second color when the ribozyme is deactivated.

12. The system of claim 10 , wherein the RNA-based masking construct is an RNA aptamer and/or comprises an RNA-tethered inhibitor.

13. The system of claim 12 , wherein the aptamer or RNA-tethered inhibitor sequesters an enzyme, wherein the enzyme generates a detectable signal upon release from the aptamer or RNA tethered inhibitor by acting upon a substrate; or
wherein the aptamer 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 RNA-tethered inhibitor inhibits an enzyme and prevents the enzyme from catalyzing generation of a detectable signal from a substrate; and
wherein the enzyme is optionally thrombin, protein C, neutrophil elastase, subtilisin, horseradish peroxidase, beta-galactosidase, or calf alkaline phosphatase or wherein the enzyme is optionally thrombin and the substrate is para-nitroanilide covalently linked to a peptide substrate for thrombin, or 7-amino-4-methylcoumarin covalently linked to a peptide substrate for thrombin.

14. The system of claim 12 , wherein the aptamer sequesters a pair of agents that when released from the aptamers combine to generate a detectable signal.

15. The system of claim 10 , wherein the one or more RNA-based masking constructs comprises an RNA oligonucleotide to which a detectable ligand and a masking component are attached; or
wherein the one or more RNA-based masking constructs comprises a nanoparticle held in aggregate by bridge molecules, wherein at least a portion of the bridge molecules comprises RNA, wherein the solution undergoes a color shift when the nanoparticle is disbursed in solution; wherein the nanoparticle is optionally a colloidal metal such as colloidal gold; or
wherein the detectable ligand is a fluorophore and the masking component is a quencher molecule.

16. The system of claim 10 , wherein the one or more RNA-based masking constructs comprises a quantum dot linked to one or more quencher molecules by a linking molecule, wherein at least a portion of the linking molecule comprises RNA.

17. The system of claim 10 , wherein the one or more RNA-based masking constructs comprises RNA in complex with an intercalating agent, wherein the intercalating agent changes absorbance upon cleavage of the RNA; wherein the intercalating agent is optionally pyronine-Y or methylene blue.

18. The system of claim 1 , wherein the one or more RNA-based masking constructs comprises a RNA-based masking construct that can be cleaved by the CRISPR effector protein having collateral cleavage activity and a RNA-based masking co

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College
• President And Fellows Harvard College

Inventors

• Zhang Feng
• Gootenberg Jonathan
• Abudayyeh Omar

Classifications

• C12N15/113 Primary

  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

• B01L3/5085

  for multiple samples, e.g. microtitration plates · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• G01N33/5308

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• B01L2300/123

  Flexible; Elastomeric · CPC title