Crispr effector system based coronavirus diagnostics

Patent metadata

Field	Value
Publication number	US-12522863-B2
Application number	US-202217731019-A
Country	US
Kind code	B2
Filing date	Apr 27, 2022
Priority date	Mar 23, 2020
Publication date	Jan 13, 2026
Grant date	Jan 13, 2026

Abstract

Systems and methods for rapid diagnostics related to the use of CRISPR effector systems and optimized guide sequences for detection of coronavirus, including multiplex lateral flow diagnostic devices and methods of use, are provided.

First claim

What is claimed is:

1 . A cartridge comprising a first and second ampoule, a lysis chamber, an amplification chamber, one or more wells, and a sample receiving chamber, wherein
a) the first ampoule fluidically is connected to the sample receiving chamber, b) the sample receiving chamber is further connected to the lysis chamber, and c) the lysis chamber is connected via a metering channel to the second ampoule and the amplification chamber, d) the one or more wells are communicatively coupled to the first ampoule and the second ampoule, wherein the first ampoule comprises a polynucleotide isolation solution capable of isolating a target polynucleotide from a crude or unprocessed sample comprising a cell or virus containing the target polynucleotide in one-step and without sample extraction, wherein the polynucleotide isolation solution comprises potassium chloride and polynucleotide binding beads, wherein the metering channel and the one or more wells are configured and sized for the flow of target polynucleotide bound beads to the amplification chamber, and wherein the second ampoule comprises one or more thermostable Cas proteins selected from SEQ ID NOs: 61644-61954; one or more-guide polynucleotides comprising a sequence capable of binding the target polynucleotide and designed to form a CRISPR-Cas complex with the one or more thermostable Cas proteins; isothermal amplification reagents comprising optimized loop-mediated isothermal amplification (LAMP) primers and amplification reagents; and a detection construct comprising a polynucleotide component, wherein, once activated by the target polynucleotide, the one or more thermostable Cas proteins cleave the polynucleotide component of the detection construct, to generate a detectable signal.

2 . The cartridge of claim 1 , wherein the polynucleotide binding beads concentrate the target polynucleotide without requiring elution of the isolated polynucleotides.

3 . A device designed to receive the cartridge of claim 1 and further comprising one or more motors connected to a plunger for rupturing of the first ampoule and the second ampoule of the cartridge and configured within the device to align with the first ampoule and the second ampoule of an inserted cartridge, a heating element configured to align with the amplification chamber of the inserted cartridge, an optical detector configured to align with the amplification chamber of the inserted cartridge, and a display.

4 . The device of claim 3 , comprising a graphical user interface for programming the device and/or for providing a readout.

5 . A system comprising a docking station and two or more devices of claim 3 , wherein the docking station is configured to receive the two or more devices.

6 . The cartridge of claim 1 , wherein the first ampoule and/or second ampoule comprise lyophilized contents.

7 . The cartridge of claim 1 , wherein the one or more thermostable Cas proteins are Brevibacillus sp. SYSU G02855 (Br) Cas12b (SEQ ID NO: 61954) or Alicyclobacillus acidiphilus (Aap) Cas 12b (SEQ ID NO: 61953).

8 . The cartridge of claim 7 , wherein the one guide polynucleotide selected from Aac guide types 1 to 5 (SEQ ID NOs: 61957-61961) or BrCas12b crRNA design 1 to 3 (SEQ ID NO: 61970-61972).

9 . The cartridge of claim 1 , wherein the optimized LAMP primers are selected from SEQ ID NOs: 1-40, 499, and 61,983-61,988.

10 . The cartridge of claim 1 , wherein the one or more guide polynucleotides are selected from SEQ ID NOs: 40,500-61,643 and SEQ ID NO: 61,989.

11 . The cartridge of claim 10 , wherein the one or more guide polynucleotides comprise a spacer specific for the N gene or S gene of SARS-COV-2.

12 . The cartridge of claim 1 , further comprising one or more additives selected from L-proline, L-histidine, b-alanine, L-serine, urea, acetamide, 4-aminobutyric acid, polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone K, 6-O-a-D-maltosyl-b-cyclodextrin, (2-hydroxypropyl)-b-cyclodextrin, a-cyclodextrin, b-cyclodextrin, methyl-b-cyclodextrin, glycine, proline, or a combination thereof.

13 . The cartridge of claim 12 , wherein the one or more additives comprise glycine, proline, or a combination thereof.

14 . The cartridge of claim 1 , wherein the one or more guide polynucleotides are optimized guide polynucleotides.

15 . A method of detecting a target polynucleotide in a sample comprising:
distributing a crude or unprocessed sample or set of samples into the sample receiving chamber of the cartridge of claim 1 , each sample comprising a cell or virus containing a target polynucleotide; incubating the sample or set of samples at conditions sufficient to allow extraction of the one or more target polynucleotides from the cell or virus, further incubating at an isothermal temperature at conditions sufficient to generate one or more amplicons of the target polynucleotides without requiring prior isolation of the target polynucleotides from the crude or unprocessed sample and the polynucleotide isolation solution; and detecting the one or more amplicons, thereby indicating one or more target polynucleotides in the sample.

16 . The cartridge of claim 1 , wherein the polynucleotide binding beads concentrate the target polynucleotides.

17 . The cartridge of claim 1 , wherein the polynucleotide binding beads are magnetic.

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology

Inventors

• Zhang Feng
• Gootenberg Jonathan
• Abudayyeh Omar
• Joung Julia
• Ladha Alim
• Altae-Tran Han
• Faure Guilhem

Classifications

• B01L3/502

  with fluid transport, e.g. in multi-compartment structures · CPC title

• C12Q1/701

  Specific hybridization probes · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• B01L2300/0816

  Cards, e.g. flat sample carriers usually with flow in two horizontal directions · CPC title