Devices for CRISPR effector system based diagnostics

Patent metadata

Field	Value
Publication number	US-11021740-B2
Application number	US-201815922837-A
Country	US
Kind code	B2
Filing date	Mar 15, 2018
Priority date	Mar 15, 2017
Publication date	Jun 1, 2021
Grant date	Jun 1, 2021

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 diagnostic device comprising one or more individual discrete volumes, each individual discrete volume comprising nucleic acid amplification reagents for amplifying a target sequence, a Cas13 protein exhibiting collateral activity and one or more guide polynucleotides comprising a guide sequence capable of binding the target sequence and designed to form a complex with the Cas protein; and a masking construct.

2. The diagnostic device of claim 1 , wherein each individual discrete volume further comprises one or more detection aptamers comprising a masked RNA polymerase promoter binding site or a masked primer binding site.

3. The diagnostic device of claim 1 , wherein the amplification reagents comprise a primer comprising an RNA polymerase promoter.

4. The diagnostic device of claim 1 , wherein the target molecule is a target DNA and each individual discrete volume further comprises a primer that binds the target DNA and comprises a RNA polymerase promoter.

5. The diagnostic device of claim 1 , wherein the one or more individual discrete volumes are droplets.

6. The diagnostic device of claim 1 , wherein the one or more individual discrete volumes are defined on a solid substrate, are spots defined on a substrate, are placed within microwells, or are microfluidic channels in a lab-on-chip (LOC) device.

7. The diagnostic device of claim 6 , wherein the substrate is a flexible materials substrate, a paper substrate, a flexible polymer based substrate, or a lateral flow strip.

8. The diagnostic device of claim 6 , wherein the LOC is a radio frequency identification (RFID) tag system.

9. The diagnostic device of claim 8 , further comprising a wireless device configured to communicate with the RFID system.

10. The diagnostic device of claim 1 , wherein the Cas13 protein is bound to each individual discrete volume.

11. The diagnostic device of claim 10 , wherein the Cas13 protein is a fusion protein comprising an affinity tag, or is linked to a biotin molecule, and wherein each individual discrete volume comprises streptavidin.

12. The diagnostic device of claim 1 , wherein the Cas13 protein comprises one or more higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains, and wherein the one or more HEPN domains comprise a RxxxxH motif sequence.

13. The diagnostic device of claim 12 , wherein the RxxxxH motif comprises a R[N/H/K]X 1 X 2 X 3 H sequence.

14. The diagnostic device of claim 13 , 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.

15. The diagnostic device of claim 1 , wherein the Cas13 protein is C2c2.

16. The diagnostic device of claim 15 , wherein the nucleic acid sequence encoding the C2c2 protein is naturally present in a prokaryotic genome within 20 kb of a Cas 1 gene.

17. The diagnostic device of claim 15 , wherein the C2c2 effector protein k from an organism of a genus 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; 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 CHKCIO04; 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.

18. The diagnostic device of claim 17 , wherein the C2c2 effector protein is a L. wadei F0279 or L. wadei F0279 (Lw2) C2c2 effector protein.

19. The diagnostic device of claim 1 , wherein the one or more guide RNAs are designed to detect a single nucleotide polymorphism in a target RNA or DNA, a splice variant of a RNA transcript, or a frameshift mutation in a target DNA or RNA.

20. The diagnostic device of claim 1 , wherein the one or more guide RNAs are designed to bind to one or more target RNA sequences that are diagnostic for a disease state.

21. The diagnostic device of claim 1 , wherein the masking construct is a RNA-based masking construct, wherein
the RNA-based masking construct is capable of suppressing generation of a detectable positive signal; or
the RNA-based masking construct is capable of masking a detectable positive signal; or
the RNA-based masking construct is capable of generating a detectable negative signal; or
the RNA-based masking construct is a conductive RNA molecule located across conductive leads; or
the RNA-based masking construct comprises a silencing RNA that is capable of suppressing generation of a gene product encoded by a reporting construct, wherein the gene product is capable of generating the detectable positive signal when expressed; or
the RNA-based masking construct is a ribozyme that is capable of generating a negative detectable signal, and wherein a positive detectable signal is generated when the ribozyme is deactivated; or
the RNA-based masking construct is a RNA aptamer and/or comprises an RNA-tethered inhibitor;
wherein the aptamer or RNA-tethered inhibitor is capable of sequestering an enzyme, wherein the enzyme is capable of generating 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 is capable of inhibiting an enzyme and prevents the enzyme from catalyzing generation of a detectable signal from a substrate or wherein the RNA-tethered inhibitor is capable of inhibiting an enzyme and prevents the enzyme from catalyzing generation of a detectable signal from a substrate; or
wherein the aptamer is capable of sequestering a pair of agents that when released from the aptamers combine to generate a detectable signal; or

wherein the RNA-based masking construct comprises a RNA oligonucleotide with a detectable ligand on a first end of the RNA oligonucleotide and a masking component on a second end of the RNA oligonucleotide;
wherein the detectable ligand is a fluorophore and the masking component is a quencher molecule.

22. The diagnostic device of claim 1 , wherein said masking construct comprises a RNA oligonucleotide designed to bind a G-quadruplex forming sequence, wherein a G-quadruplex structure is formed by the G-quadruplex forming sequence upon cleavage of the masking construct, and wherein the G-quadruplex structure is capable of generating a detectable positive signal.

23. The diagnostic device of claim 1 , wherein said masking construct comprises a first molecule and a second molecule and a lateral flow strip comprises two capture lines with an anti-first molecule antibody hybridized at a first capture line and an anti-second molecule antibody hybridized at a second capture line.

24. The diagnostic device of claim 23 , wherein the first molecule is FITC, and the second molecule is biotin.

25. The diagnostic device of claim

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College

Inventors

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

Classifications

• C12N15/11 Primary

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• G01N33/54388

  based on lateral flow · CPC title

• C12Q1/6816

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

• B01L2300/0825

  Test strips · CPC title

• C12N9/22

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