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US-12618101-B2 - Sherlock assays for tick-borne diseases

US12618101B2US 12618101 B2US12618101 B2US 12618101B2US-12618101-B2

Abstract

Provided herein is a nucleic acid detection system comprising a detection CRISPR system having an effector protein and one or more guide RNAs each designed to bind to corresponding target molecules that are diagnostic for a tick-borne disease state; and an RNA-based masking construct. In some embodiments, the detection system of may comprise i) two or more CRISPR systems, each CRISPR system comprising an effector protein and a guide RNA designed to bind to a corresponding target molecule that is diagnostic for a tick-borne disease state; and ii) a set of detection constructs, each detection construct comprising a cutting motif sequence that is preferentially cut by one of the activated CRISPR effector proteins. Exemplary tick-borne detectable microbes include Babesia microti, Anaplasma phagocytophilum , and Borrelia miyamotoi.

Inventors

  • Pardis Sabeti
  • Jacob LEMIEUX
  • Anne PIANTADOSI
  • Erica NORMANDIN
  • Gordon Adams
  • Eric Rosenberg

Assignees

  • THE BROAD INSTITUTE, INC.
  • PRESIDENT AND FELLOWS OF HARVARD COLLEGE
  • THE GENERAL HOSPITAL CORPORATION

Dates

Publication Date
20260505
Application Date
20200313

Claims (20)

  1. 1 . A nucleic acid detection system comprising: a. a CRISPR system comprising an effector protein and one or more guide RNAs comprising sequences selected from SEQ ID NOs: 6-13 and 15-19; and b. an RNA-based masking construct, wherein the one or more guide RNAs bind to one or more target nucleotide sequences, wherein the one or more guide RNAs detect a tick-borne disease state, wherein the tick-borne disease state is babesiosis.
  2. 2 . A nucleic acid detection system, comprising: i) two or more CRISPR systems, each CRISPR system comprising a CRISPR effector protein and a guide RNA comprising sequences selected from SEQ ID NOs: 6-13 and 15-19; and ii) a set of detection constructs, each detection construct comprising a cutting motif sequence that is cut by one of the CRISPR effector proteins wherein the guide RNA binds to a target nucleotide sequence, wherein the guide RNA detects a tick-borne disease state, wherein the tick-borne disease state is babesiosis.
  3. 3 . The detection system of claim 1 , wherein the one or more target nucleotide sequences are derived from Babesia microti.
  4. 4 . The detection system of claim 1 , wherein the one or more guide RNAs bind to the cytB region of Babesia microti.
  5. 5 . The detection system of claim 1 , wherein the one or more guide RNAs bind to variants of Babesia microti.
  6. 6 . The detection system of claim 1 , wherein the one or more guide RNAs comprise 95% sequence identity to nucleotides of SEQ ID NOs: 6-13 and 15-19.
  7. 7 . The nucleic acid detection system of claim 1 , further comprising one or more nucleic acid amplification reagents.
  8. 8 . The nucleic acid detection system of claim 1 , wherein the one or more target nucleotide sequences are a target DNA sequence.
  9. 9 . The nucleic acid detection system of claim 1 , wherein the one or more target nucleotide sequences comprises an SNP.
  10. 10 . The nucleic acid detection system of claim 9 , wherein the one or more guide RNAs bind to the one or more target nucleotide sequences associated with the tick-borne disease state at a SNP cytB M134I of B. microti.
  11. 11 . A lateral flow device comprising the nucleic acid detection system of claim 1 .
  12. 12 . The lateral flow device of claim 11 , wherein the CRISPR system is freeze-dried on a lateral flow strip.
  13. 13 . The lateral flow device of claim 11 , wherein the lateral flow device comprises a substrate comprising a first end, wherein the first end comprises a sample loading portion and a first region loaded with a detectable ligand, the nucleic acid detection system, a first capture region comprising a first binding agent, and a second capture region comprising a second binding agent, optionally wherein the sample loading portion comprises a receiving input for a blood stick.
  14. 14 . The lateral flow device of claim 13 , wherein the sample loading portion further comprises one or more amplification reagents to amplify the one or more target nucleotide sequences, wherein the reagents optionally comprise reagents for nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), PCR, multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), or ramification amplification method (RAM).
  15. 15 . The lateral flow device of claim 11 , wherein the RNA construct comprises a first molecule on a first end and a second molecule on a second end, optionally wherein a first capture region comprises a first binding agent that specifically binds the first molecule of a reporter construct.
  16. 16 . The lateral flow device of claim 15 , wherein the first molecule is FITC and the second molecule is biotin, or vice versa.
  17. 17 . The lateral flow device of claim 13 , wherein the first binding agent is an antibody that is fixed or otherwise immobilized to the first capture region, or wherein the second capture region comprises a second binding agent that specifically binds a second molecule of a reporter construct, or the detectable ligand.
  18. 18 . The lateral flow device of claim 17 , wherein the second binding agent is an antibody or an antibody-binding protein that is fixed or otherwise immobilized to the second capture region.
  19. 19 . A method for detecting target nucleic acids in a sample, comprising: distributing a sample or set of samples into one or more individual discrete volumes, the individual discrete volumes comprising the nucleic acid detection system of claim 1 .
  20. 20 . The method of claim 19 , wherein the sample is blood, a red blood cell supernatant, plasma, or cerebrospinal fluid.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is the U.S. National Stage of International Application No. PCT/US2020/022776, filed Mar. 13, 2020, which claims the benefit of U.S. Provisional Application No. 62/818,739 filed Mar. 14, 2019 and U.S. Provisional Application 62/860,225 filed Jun. 11, 2019. The entire contents of the above-identified applications are hereby fully incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant No. D18AC00006 awarded by the Department of Defense. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (BROD-4100US_ST25.txt”; Size is 11,919 bytes and it was created on Nov. 5, 2024) is herein incorporated by reference in its entirety. TECHNICAL FIELD The subject matter disclosed herein is generally directed to rapid diagnostics related to the use of CRISPR effector systems. BACKGROUND Tick-borne diseases (TBD) such as Lyme disease (LD), babesiosis, and anaplasmosis have emerged over the past 40 years as a major threat to public health. Borrelia burgdorferi, the causative pathogen for LD, and co-infections are increasing in prevalence and expanding their geographic range. Poorly performing diagnostic assays have hampered our ability to detect and understand these pathogens. This is particularly true for LD, for which diagnostic tests are insensitive in acute infection and unreliable for LD-causing organisms outside of the United States (Branda et al. Clin Infect Dis 57 (3): 333-340 (2013); Makhani et al. J Clin Microbiol 49 (1): 455-457 (2011)). The tick that transmits LD in the US, Ixodes scapularis, also transmits several other important tick-borne infections. Babesia microti and Anaplasma phagocytophilum infections can lead to shock and respiratory failure (Vannier et al. NEJM 366 (25): 2397-2407 (2012)). Powassan virus (POWV) infection can result in an often-fatal encephalitis (Ebel Annu Rev Entomol 55:95-110 (2010)). New tick-borne pathogens continue to be discovered or recognized, including Bourbon virus (Kosoy et al. Emerg Infect Dis 21 (5): 760-764 (2015)), Heartland virus (McMullan et al. NEJM 367 (9): 834-841 (2012)), Borrelia miyamotoi (Krause et al. nejm.org/doi/full/10.1056/NEJMc1215469 (2013); Gugliotta et al. NEJM 368 (3): 240-245 (2013)), Borrelia mayonii (Pritt et al. Lancet Infect Dis sciencedirect.com/science/article/pii/S1473309915004648 (2016)), and the Ehrlichia muris-like agent (Pritt et al. NEJM 365 (5): 422-429 (2011)). These infections can cause severe disease, but currently only limited information is understood about their pathogenesis. Powassan virus, an emerging tick-borne flavivirus causes severe encephalitis and is transmitted by Ixodes scapularis ticks, with little known about strains that cause human infection. Two major problems need to be addressed in the field of TBD. The first problem is insensitive diagnostics. Current serological testing for LD is unreliable, performs poorly in early infection, does not consistently distinguish between acute and prior infection or between strains, and cannot be used with confidence in immunocompromised individuals. More sensitive diagnostics are needed; these should be cheap, field deployable, and multiplexed, since tick-borne pathogens are frequently encountered as co-infections. The second major problem is a lack of sequence- and strain-specific diagnostics and an incomplete understanding of the role of pathogen genetics in influencing clinical disease. Sequence-based diagnostics that identify the infective strain are needed. This is crucial in elucidating the pathogenic basis of heterogeneity in clinical manifestations of tick-borne disease. It would be useful to know why some patients experience severe central nervous system (CNS) disease or Lyme arthritis, and some experience only erythema migraines, whereas others have no rash at all. These patterns may be mediated by pathogen genes. There is a critical need for rapid, sensitive, sequence-specific, point-of-care (POC) diagnostics to guide treatment of TBD. SUMMARY In certain example embodiments, the invention provides a nucleic acid detection system comprising a detection CRISPR system having an effector protein and one or more guide RNAs each designed to bind to corresponding target molecules that are diagnostic for a tick-borne disease state; and an RNA-based masking construct. In some embodiments, the detection system may comprise i) two or more CRISPR systems, each CRISPR system comprising an effector protein and a guide RNA designed to bind to a corresponding target molecule that is diagnostic for a tick-borne disease state; and ii) a set of detection constructs, each detection construct comprising a cutting motif sequence that is preferentially cut by one of the activated CRISPR effector proteins. In some embodiments, guide RNAs may be designed to bind to Bab