CRISPR effector system based diagnostics for malaria detection

Patent metadata

Field	Value
Publication number	US-11618928-B2
Application number	US-201815952132-A
Country	US
Kind code	B2
Filing date	Apr 12, 2018
Priority date	Apr 12, 2017
Publication date	Apr 4, 2023
Grant date	Apr 4, 2023

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 nucleic acid detection system comprising:
a CRISPR system comprising a Type VI Cas effector protein exhibiting collateral activity and one or more guide RNAs capable of binding one or more target nucleic acid sequences of a mosquito-borne parasite, each guide RNA comprising a guide sequence capable of hybridizing to a complementary target nucleic acid sequence of a mosquito-borne parasite and subsequently forming a CRISPR complex of the Type VI Cas effector protein with each guide RNA comprising the guide sequence hybridized to the complementary target nucleic acid sequence; and
an RNA-based masking construct.

2. The detection system of claim 1 , wherein the mosquito-borne parasite is selected from the genus Plasmodium.

3. The detection system of claim 2 , wherein the Plasmodium is selected from the species Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae , and Plasmodium knowlesi.

4. The detection system of claim 1 , wherein the one or more target nucleic acid sequences are DNA or RNA and wherein the one or more target nucleic acid sequences can be used for species or strain identification.

5. The detection system of claim 4 , wherein
when the one or more target nucleic acid sequences are DNA sequences, the DNA sequences are selected from gene sequences that are conserved within species but divergent between species, and
when the one or more target nucleic acid sequence are RNA sequences, the RNA sequences are selected from transcripts of said gene sequences that are conserved within species but divergent between species.

6. The detection system of claim 4 , wherein
when the one or more target nucleic acid sequences are DNA, the DNA is selected from highly expressed gene sequences, highly conserved gene sequences, gene sequences that are present in multiple copies in the genome, and combinations thereof; and
when the target nucleic acid sequence is RNA, the RNA is selected from transcripts of said highly expressed gene sequences, transcripts of said highly conserved gene sequences, transcripts of said gene sequences that are present in multiple copies in the genome, and combinations thereof.

7. The detection system of claim 4 , wherein the one or more target nucleic acid sequences are selected from a transcript of a ribosomal gene cytochrome b (CYTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Histone H2B, enolase, and lactate dehydrogenase (LDH).

8. The detection system of claim 7 , wherein the transcript of a ribosomal gene is an 18S ribosomal DNA transcript.

9. The detection system of claim 4 , wherein the one or more target nucleic acid sequences can be used for the detection of one or more parasite drug-resistance markers; and wherein the drug is selected from aryl-amino alcohols, antifolate drugs, pyrimethamine, atemisins drugs, proguanil, the combination of atovaquone and proguanil, and combinations thereof.

10. The detection system of claim 9 , wherein the aryl-amino alcohols are selected from quinine, quinine derivatives, lipophilic hydroxynapthoquinone, and combinations thereof.

11. The detection system of claim 10 , wherein the quinine derivatives are selected from chloroquine, amodiaquine, mefloquine, piperaquine, lumefantrine, primaquine, and combinations thereof.

12. The detection system of claim 10 , wherein the lipophilic hydroxynaphthoquinone analog is atovaquone.

13. The detection system of claim 9 , wherein the antifolate drugs are selected from sulfa drugs.

14. The detection system of claim 13 , wherein the sulfa drugs are selected from sulfadoxine, dapsone, and combinations thereof.

15. The detection system of claim 4 , wherein the one or more target nucleic acid sequences are selected from sequences encoding transporter proteins and sequences encoding proteins involved in the translocation of protons across the inner mitochondrial membrane.

16. The detection system of claim 15 , wherein the transporter proteins are selected from a protein from the drug/metabolite transporter family, the ATP-binding cassette (ABC) protein involved in substrate translocation, and combinations thereof.

17. The detection system of claim 16 , wherein the ATP-binding cassette (ABC) protein involved in substrate translocation is selected from the ABC transporter C subfamily, the Na+/H+ exchanger, and combinations thereof.

18. The detection system of claim 15 , wherein the proteins involved in the folate pathway are selected from the dihydropteroate synthase, the dihydrofolate reductase activity, the dihydrofolate reductase-thymidylate synthase, and combinations thereof.

19. The detection system of claim 15 , wherein the one or more target nucleic acid sequences comprise a mitochondrial gene sequence.

20. The detection system of claim 19 , wherein the protein involved in the translocation of protons across the inner mitochondrial membrane is the cytochrome b complex.

21. The detection system of claim 4 , wherein the one or more target nucleic acid sequences are selected from the P. falciparum chloroquine resistance transporter gene (pfcrt), the P. falciparum multidrug resistance transporter I (pfmdr1), the P. falciparum multidrug resistance-associated protein gene (Pfmrp), the P. falciparum Na+/H+ exchanger gene (pfnhe), the P. falciparum Ca2+ transporting ATPase 6 (pfatp6) gene, the P. falciparum dihydropteroate synthase (pfdhps) gene, the P. falciparum dihydrofolate reductase activity (pfdhpr) gene, the P. falciparum dihydrofolate reductase-thymidylate synthase (pfdhfr) gene, the cytochrome b gene, and functional heterologous genes thereof in other Plasmodium species.

22. The detection system of claim 21 , wherein the target sequence is selected from pfcrt, pfmdr1, pfdhjr, Cytochrome b, gtp cyclohydrolase, and Kelch13.

23. A method for detecting a mosquito-borne parasite by detecting one or more target nucleic acids in one or more samples, 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 ;
incubating the sample or set of samples under conditions sufficient to allow binding of the one or more guide RNAs to the one or more target nucleic acid sequences;
activating the Type VI Cas effector protein via binding of the one or more guide RNAs to the one or more target nucleic acid sequences, wherein activating the Type VI Cas effector protein results in modification of the RNA-based masking construct such that a detectable positive signal is generated; and
detecting the detectable positive signal, wherein detecting the detectable positive signal indicates a presence of one or more target nucleic acids in the sample.

24. The method of claim 23 , wherein the one or more target nucleic acid sequences are a one or more target DNA sequences and the method further comprises binding the one or more target DNA sequences with a primer comprising an RNA polymerase site.

25. A polypeptide detection system comprising:
a CRISPR system comprising a Type VI Cas effector protein and one or more guide RNAs capable of binding one or more trigger RNAs of a mosquito-borne parasite, each guide RNA comprising a guide sequence capable of hybridizing with a complementary trigger RNA sequence and subsequently forming a CRISPR complex of the Type VI Cas effector protein with each guide RNA comprising the guide sequence hybrid

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College

Inventors

• Abudayyeh Omar
• Collins James Joseph
• Gootenberg Jonathan
• Zhang Feng
• Lander Eric S
• Neafsey Daniel
• Early Angela

Classifications

• C12N2310/3517

  Marker; Tag · CPC title

• C12N2310/16

  Aptamers · CPC title

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title

• C12N9/22

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

• C12N2310/3519

  Fusion with another nucleic acid · CPC title