CRISPR effector system based diagnostics

• US 10,266,886 B2
• Filed: 03/09/2018
• Issued: 04/23/2019
• Est. Priority Date: 12/09/2016
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Abudayyeh, Omar, Collins, James Joseph, Gootenberg, Jonathan, Zhang, Feng, Lander, Eric S.
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US15/917,546
• Publication Number:
  US 20180340218A1
• Time in Patent Office: 410 Days
• Field of Search: None
• CPC Class Codes: C12N 15/113 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 Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: G01N 33/00 Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

1. A system for detecting the presence of a nucleic acid target sequence in an in vitro sample, comprising:

reagents for amplifying the target sequence;

a Cas13;

at least one guide polynucleotide comprising a guide sequence that hybridizes with the target sequence, and designed to form a complex with the Cas13; and

an RNA-based masking construct comprising a non-target sequence; and

wherein the Cas13 exhibits collateral RNase activity and cleaves the non-target sequence of the RNA-based masking construct once activated by the target sequence.

2. The system of claim 1, wherein the target sequence is a target DNA sequence and the system further comprises an RNA polymerase and a primer designed to bind the target DNA sequence and further comprises an RNA polymerase promoter.

3. The system of claim 1, wherein the Cas13 comprises one or more HEPN domains.

4. The system of claim 3, wherein the HEPN domain comprise a RxxxxH motif sequence.

5. The system of claim 4, wherein the RxxxxH motif comprises a R[N/H/K]X₁X₂X₃H sequence, 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.

6. The system of claim 1, wherein the Cas13 is a Cas13a, a Cas13b, or a Cas13c.

7. The system of claim 1, wherein the reagents for amplifying the one or more target sequences are isothermal amplification reagents.

8. The system of claim 7, wherein the isothermal amplification reagents are nucleic-acid sequence-based amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification (HDA), or nicking enzyme amplification.

9. A system for detecting the presence of one or more target polypeptides in an in vitro sample comprising:

an RNA-based masking construct comprising a non-target sequence; and

one or more detection aptamers, each designed to bind to one of the one or more target polypeptides, and each detection aptamer comprising a masked RNA polymerase promoter binding site or a masked primer binding site and a trigger sequence template, encoding a trigger sequence;

a Cas13

at least one guide polynucleotide comprising a guide sequence that hybridizes with the trigger sequence encoded by the trigger sequence template; and

wherein the Cas13 exhibits collateral RNase activity and cleaves the non-target sequence of the RNA-based masking construct once activated by the trigger sequence.

10. The system of claim 9, further comprising nucleic acid amplification reagents to amplify the trigger sequence.

11. The system of claim 10, wherein the nucleic acid amplification reagents to amplify the trigger sequence are isothermal amplification reagents.

12. The system of claim 11, wherein the isothermal amplification reagents are nucleic-acid sequence-based amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification, or nicking enzyme amplification.

13. The system of claim 9, wherein the Cas13 comprises one or more higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains.

14. The system of claim 13, wherein the HEPN domain comprise a RxxxxH motif sequence.

15. The system of claim 14, wherein the RxxxxH motif comprises a R[N/H/K]X₁X₂X₃H sequence, 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.

16. The system of claim 9, wherein the Cas13 is a Cas13a, a Cas13b, or a Cas13c.