Granted Patent B2

US 12,618,101 · App. 17/439,357 · Granted May 5, 2026

Inventors: Pardis Sabeti (Cambridge, MA); Jacob Lemieux (Boston, MA); Anne Piantadosi (Boston, MA); Erica Normandin (Cambridge, MA); Gordon Adams (Cambridge, MA); Eric Rosenberg (Boston, MA)

Assignees: The Broad Institute, Inc.; President and Fellows of Harvard College; The General Hospital Corporation

C12Q1/6844B01L3/5023C12Q1/689B01L2300/069B01L2300/0825

Abstract

Claims (35)

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 . 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 . The detection system of claim 1 , wherein the one or more target nucleotide sequences are derived from Babesia microti.

4 . The detection system of claim 1 , wherein the one or more guide RNAs bind to the cytB region of Babesia microti.

5 . The detection system of claim 1 , wherein the one or more guide RNAs bind to variants of Babesia microti.

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 . The nucleic acid detection system of claim 1 , further comprising one or more nucleic acid amplification reagents.

8 . The nucleic acid detection system of claim 1 , wherein the one or more target nucleotide sequences are a target DNA sequence.

9 . The nucleic acid detection system of claim 1 , wherein the one or more target nucleotide sequences comprises an SNP.

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 . A lateral flow device comprising the nucleic acid detection system of claim 1 .

12 . The lateral flow device of claim 11 , wherein the CRISPR system is freeze-dried on a lateral flow strip.

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 . 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 . 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 . The lateral flow device of claim 15 , wherein the first molecule is FITC and the second molecule is biotin, or vice versa.

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 . 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 . 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 . The method of claim 19 , wherein the sample is blood, a red blood cell supernatant, plasma, or cerebrospinal fluid.

21 . The method of claim 19 , wherein the target nucleic acid is from a sample of cell free DNA.

22 . The method of claim 19 , wherein the target nucleic acid is DNA and wherein the method further comprises extracting DNA from cells in the sample.

23 . The method of claim 19 , wherein the sample is collected on a nucleic acid collection card, optionally further comprising eluting the sample from the nucleic acid collection card.

24 . The nucleic acid detection system of claim 2 , wherein each CRISPR system further comprises an activation sequence, wherein the CRISPR effector protein is activated upon cleavage of the activation sequence.

Assignments (7)

Continuity (3)

References Cited (103)