Crispr effector system based coronavirus diagnostics
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.
1. A single reaction composition for detecting the presence of a target polynucleotide in a crude or unprocessed sample, comprising:
a DNA isolation solution capable of isolating target RNA from the crude or unprocessed sample comprising a cell or virus containing a target RNA;
one or more thermostable Cas proteins possessing collateral activity;
at least one guide polynucleotide comprising a sequence capable of binding the target RNA and designed to form a CRISPR-Cas complex with the one or more 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 the one or more Cas proteins exhibit collateral nuclease activity and cleave the polynucleotide component of the detection construct once activated by the target RNA, thereby generating a detectable signal.
2. The composition of claim 1 , wherein at least one of the one or more Cas proteins is a Type V Cas.
3. The composition of claim 2 , wherein the at least one of the one or more Cas proteins is a Cas12b is selected from the group consisting of SEQ ID NOs: 61644-61954.
4. The composition of claim 3 , wherein the Cas12b is Brevibacillus sp. SYSU G02855 (Br) Cas12b or Alicyclobacillus acidiphilus (Aap) Cas 12b.
5. The composition of claim 4 , wherein the at least one guide polynucleotide comprises a sequence selected from SEQ ID NOs: 61957-61961 and 61970-61972.
6. The composition of claim 1 , wherein the optimized LAMP primers are selected from SEQ ID NOs: 1-40, 499 and 61,983-61,988.
7. The composition of claim 1 , wherein the at least one guide polynucleotide is selected from SEQ ID NOs: 40,500-61,643 and SEQ ID NO: 61,989.
8. The composition of claim 7 , wherein the at least one guide polynucleotide comprises a spacer specific for the N gene or S gene of SARS-CoV-2.
9. The composition of claim 1 , further comprising one or more additional additives selected from the group consisting of glycine, taurine, histidine, and combinations thereof.
10. The composition of claim 1 , further comprising RNA binding beads.
11. The composition of claim 1 , wherein the at least one guide polynucleotide is an optimized guide polynucleotide.
12. The composition of claim 1 , further comprising reverse transcriptase.
13. A method for detecting coronavirus in a sample, the method comprising;
distributing a crude or unprocessed sample comprising a cell or virus containing a target RNA or set of said samples into individual discrete volumes, each individual discrete volume comprising the composition of claim 1 ;
incubating the crude or unprocessed sample or set of said samples in the composition of claim 1 at conditions sufficient to isolate the target RNA of the cell or virus via reagents of the DNA isolation solution;
amplifying the isolated target RNA using isothermal amplification, wherein, optionally, isolation of the target RNA from the composition of claim 1 between the incubating and amplifying steps is omitted; and
detecting amplified target RNA by binding of the CRISPR-Cas complex to the amplified target RNA, wherein binding of the amplified target RNA activates cleavage of the detection construct thereby generating a detectable signal.