Method and kit for detection of polynucleotide
The invention describes a kit for detection of a target polynucleotide using a CRISPR effector system that comprises CAS9 from Francisella novicida , a synthetic sgRNA and a detection scheme based on binding and subsequent enzymatic cleavage of the target polynucleotide. The invention also describes a method for detection of a target polynucleotide using the kit. The kit can be applied to both pathogenic and non-pathogenic polynucleotides and can be used to distinguish polynucleotides different by a single mismatch without the need for sequencing. The kit can also be used for detection of COVID-19. The kit is economical, easy to assemble and provides a robust and rapid readout that can be appropriately adapted for point of care applications.
1 . A kit for detecting a target polynucleotide, the kit comprising:
a. reverse and forward primers;
b. dNTPS;
c. a PCR buffer;
d. a DNA polymerase;
e. a FnCas9 protein having SEQ ID NO. 2;
f. a single guide RNA; and
g. a CRISPR-CAS9 reaction buffer.
2 . The kit of claim 1 , wherein the single guide RNA has a sequence selected from the group consisting of SEQ ID NOS.: 32, 33, 36, 39, 42, 45, 48, 51-64, 67, 76, 77, 79, and 80.
3 . The kit of claim 1 , wherein the primers are selected from sequences having SEQ ID NOS.: 37, 38, 40, 41, 43, 44, 46, 47, 49, 50, 65, 66, 68, or 69.
4 . The kit of claim 1 , wherein the PCR buffer comprises Recombinase Polymerase Amplification (RPA) Rehydration buffer and Magnesium Acetate.
5 . The kit of claim 1 , wherein the kit further comprises reverse transcriptase.
6 . The kit of claim 1 , wherein the CRISPR-CAS9 reaction buffer comprises one or more of HEPES, KCl, DTT, glycerol and MgCl 2 .
7 . The kit of claim 1 , wherein the primers are labelled with biotin.
8 . The kit of claim 1 , wherein the FnCas9 protein is complexed with a single guide RNA and labelled with a chemical.
9 . The kit of claim 1 , wherein the FnCas9 protein is isolated from Francisella novicida.
10 . The kit of claim 1 , wherein the FnCas9 protein is coupled with a fluorophore tag and has SEQ ID NO: 3.
11 . The kit of claim 1 , wherein the kit further comprises a reaction tube, a dipstick buffer and a paper strip.
12 . The kit of claim 11 , wherein the dipstick buffer is a Tris buffered saline.
13 . The kit of claim 11 , wherein the paper strip is made of a nitrocellulose membrane coated with a biotin-ligand and polyclonal (goat) digoxigenin antibody or polyclonal (rabbit) anti-FITC antibody in gold conjugate.
14 . A FnCas9 ribonucleoprotein complex comprising a Cas9 protein from Francisella novicida having the amino acid sequence as set forth in SEQ ID NO. 2, and a single guide RNA having SEQ ID NO. 79.
15 . The FnCas9 ribonucleoprotein complex of claim 14 , wherein the single guide RNA or its corresponding sequence is selected from the group consisting of SEQ ID NOS.: 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 19-36, 39, 42, 45, 48, 76, 77, 79, 80.
16 . The FnCas9 ribonucleoprotein complex of claim 14 , wherein the corresponding DNA targeting region in SARS-CoV2 has a sequence as set forth in SEQ ID NOS.: 64, 67.