I/We Claim:
1. A recombinant expression vector comprising:
(a) a guide polynucleotide complementary to a target sequence, operably linked to a promoter, wherein the target sequence has a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and wherein the guide polynucleotide has contiguous nucleotides complementary to the target sequence in the range of 24-30 nucleotides, and wherein the guide polynucleotide is complementary to a region within at least 50 nucleotides downstream of a canonical stop codon or a pre-mature stop codon of the target sequence; and
(b) a polynucleotide encoding dCas13 protein having a nucleotide sequence selected from SEQ ID NO:5, and SEQ ID NO: 31, operably linked to a promoter.
2. The recombinant expression vector as claimed in claim 1, wherein the guide polynucleotide has contiguous nucleotides complementary to the target sequence in the range of 26-28 nucleotides.
3. The recombinant expression vector as claimed in claim 1 or 2, wherein the guide polynucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 30.
4. The recombinant expression vector as claimed in claim 1, wherein the promoter driving the expression of the guide polynucleotide is selected from the group consisting of U6 promoter, tRNAVal promoter, and H1 promoter.
5. The recombinant expression vector as claimed in claim 1, wherein the promoter driving the expression of dCas13 protein is selected from the group consisting of chicken β-actin promoter, SV40 promoter, CMV promoter, Ubc promoter, and CAG promoter.
6. A bacterial host cell comprising the recombinant expression vector as claimed in anyone of the claims 1-5.
7. A method of inducing translational readthrough across a canonical stop codon or a pre-mature stop codon of a target sequence, said method comprising:
(a) obtaining a human cell comprising a polynucleotide having a canonical stop codon or a pre-mature stop codon of a target sequence, wherein the target sequence has a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9;
(b) obtaining a recombinant expression vector of claim 1, wherein the recombinant expression vector comprising: (i) a guide polynucleotide complementary to a target sequence, operably linked to a promoter, wherein the target sequence has a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and wherein the guide polynucleotide has contiguous nucleotides complementary to the target sequence in the range of 24-30 nucleotides, and wherein the guide polynucleotide complementary to a region within at least 50 nucleotides downstream of a canonical stop codon or a pre-mature stop codon of the target sequence; and (ii) a polynucleotide encoding dCas13 protein having a nucleotide sequence selected from SEQ ID NO: 5, and SEQ ID NO: 31, operably linked to a promoter;
(c) transfecting the human cell with the recombinant expression vector to obtain a transfected cell, wherein the transfected cell produces guide RNA/ dCas13 complex comprising the dCas13 protein complexed with the guide RNA, and wherein the guide RNA is capable of hybridizing to the target sequence, and wherein the guide RNA recruits the dCas13 protein hybridizing proximally downstream to the canonical stop codon or pre-mature stop codon of the target sequence; and
(d) analysing the expression levels of target proteins in the transfected cells,
wherein an increase in the levels of the target proteins in the transfected
cells indicates an increased translational readthrough across the canonical
stop codon or the pre-mature stop codon of the target sequence.
8. A method of inducing translational readthrough across pre-mature stop codon
of a target haemoglobin subunit beta (HBB) gene, said method comprising:
(a) obtaining a human cell having a pre-mature stop codon in HBB gene, wherein the HBB gene has a nucleotide sequence as set forth in SEQ ID NO: 9;
(b) obtaining a recombinant expression vector comprising: (i) a guide polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, complementary to a region downstream of a pre-mature stop codon in HBB gene having a nucleotide sequence as set forth in SEQ NO: 9, operably linked to a promoter; and (ii) a polynucleotide encoding a dCas13a protein, having a nucleotide sequence as set forth in SEQ ID NO: 5, operably linked to a promoter;
(c) transfecting the human cell with the recombinant expression vector to obtain a transfected cell, wherein the transfected cell produces guide RNA/ dCas13a complex comprising the dCas13a protein complexed with the guide RNA, and wherein the guide RNA is capable of hybridizing to the target haemoglobin subunit beta (HBB) gene, and wherein the guide RNA recruits the dCas13a protein hybridizing proximally downstream to the pre-mature stop codon in HBB gene having a nucleotide sequence as set forth in SEQ NO: 9; and
(d) analysing the expression levels of a β-globin protein in the transfected cells, wherein an increase in the expression levels of β-globin protein in the transfected cell indicates an increased translational readthrough across the pre-mature stop codon of the HBB gene to produce the β-
globin protein. 9. A method of inducing translational readthrough across canonical stop codon of a target polynucleotide encoding Argonaute 1 (Ago1) protein, said method comprising:
(a) obtaining a human cell comprising a AGO1 target polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 6, wherein the polynucleotide has a canonical stop codon;
(b) obtaining a recombinant expression vector comprising: (i) a guide polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, complementary to a region downstream of canonical stop codon of the AGO1 target polynucleotide sequence as set forth in SEQ NO: 6, operably linked to a promoter; and (ii) a polynucleotide encoding a dCas13a protein, having a nucleotide sequence as set forth in SEQ ID NO: 5, operably linked to a promoter;
(c) transfecting the human cell with the recombinant expression vector to obtain transfected cells, wherein the transfected cell produces guide RNA/ dCas13a complex comprising the dCas13a protein complexed with the guide RNA, and wherein the guide RNA is capable of hybridizing to the AGO1 target polynucleotide sequence, and wherein the guide RNA recruits the dCas13a protein hybridizing proximally downstream to the canonical stop codon of SEQ ID NO: 6; and
(d) analysing the expression levels of a target protein Ago1x in the transfected cells, wherein an increase in the expression levels of the Ago1x protein in the transfected cells indicates an increased translational readthrough across a canonical stop codon of the polynucleotide encoding Argonaute 1 (Ago1) protein. 10. A method of inducing translational readthrough across canonical stop codon of a polynucleotide encoding Vascular endothelial growth factor (VEGF-A), said method comprising:
(a) obtaining a human cell comprising a polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 8, wherein the polynucleotide has a canonical stop codon;
(b) obtaining a recombinant expression vector comprising: (i) a guide polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, complementary to a region downstream of a canonical stop codon of the VEGF-A target polynucleotide sequence as set forth in SEQ ID NO: 8, operably linked to a promoter; and (ii) a polynucleotide encoding a dCas13a protein, having a nucleotide sequence as set forth in SEQ ID NO: 5, operably linked to a promoter,
(c) transfecting the human cell with the recombinant expression vector to obtain transfected cells, wherein the transfected cell produces guide RNA/ dCas13a complex comprising the dCas13a protein complexed with the guide RNA, and wherein the guide RNA is capable of hybridizing to the VEGF-A target polynucleotide sequence, and wherein the guide RNA recruits the dCas13a protein hybridizing proximally downstream to the canonical stop codon of SEQ ID NO: 8; and
(d) analysing the expression levels of a target protein VEGF-Ax in the transfected cells, wherein an increase in the expression levels of the VEGF-Ax protein in the transfected cells indicates an increased translational readthrough across a canonical stop codon of a polynucleotide encoding VEGF-A protein.
11. A method of inducing translational readthrough across canonical stop codon of a target polynucleotide encoding Mitochondrial carrier homolog 2 protein·(MTCH2), said method comprising:
(a) obtaining a human cell comprising a polynucleotide having nucleotide sequence as set forth in SEQ NO: 7, wherein the polynucleotide has a canonical stop codon;
(b) obtaining a recombinant expression vector comprising: (i) a guide polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, complementary to a region downstream of canonical stop codon of MTCH2 target polynucleotide sequence as set forth in SEQ ID NO: 7, operably linked to a promoter; and (ii) a polynucleotide encoding a dCas13a protein, having a nucleotide sequence as set forth in SEQ ID NO: 5, operably linked to a promoter;
(c) transfecting the human cell with the recombinant expression vector to obtain transfected cells, wherein the transfected cell produces guide RNA/ dCas13a complex comprising the dCas13a protein complexed with the guide RNA, and wherein the guide RNA is capable of hybridizing to the MTCH2 target polynucleotide sequence, and wherein the guide RNA recruits the dCas13a protein hybridizing proximally downstream to the canonical stop codon of SEQ NO: 7; and
(d) analysing the expression levels of readthrough proteins of MTCH2 in the transfected cells, wherein an increase in the expression levels of the readthrough proteins of MTCH2 in the transfected cells indicates an increased translational readthrough across a canonical stop codon of the target polynucleotide encoding MTCH2 protein.
12. The method as claimed in anyone of the claims 7-11, wherein the human cell is selected from the group consisting of HEK293, HeLa, HepG2, MCF7, HPMEC, huh7, U2OS, and K562 cells.
13. The method as claimed in claim in claim 8, wherein the human cell has at least one non-sense mutation at a nucleotide position in SEQ ID NO: 9, and wherein the non-sense mutation at the nucleotide position is selected from the group consisting of positions at 7, 8, 16, 18, 23, 27, 36, 38, 40, 44, 44, 60, 62, 83, 91, 96, 113, 122, 128, 131, 133, 145, and 146.
14. The method as claimed in claim in claim 7, wherein the guide polynucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 30.
15. The method as claimed in anyone of the claims 7-11, wherein transfecting the human cell is done by a method selected from the group consisting of lipofection, nucleofection, electroporation, microinjection, and viral delivery systems.
16. The method as claimed in anyone of the claims 7-11, analysing the expression levels of proteins is done by a method selected from the group consisting of western blotting, ELISA, mass spectrometry, luminescence-based reporter assays, and fluorescence-based reporter assays.
17. The method as claimed in claim 7, wherein the promoter driving the expression of the guide polynucleotide is selected from the group consisting of U6 promoter, tRNAVal promoter, and H1 promoter, and wherein the promoter driving the expression of dCas13 protein is selected from the group consisting of chicken β-actin promoter, SV40 promoter, CMV promoter, Ubc promoter, EF-1α, and CAG promoter.
18. The method as claimed in anyone of the claims 8-11, wherein the promoter driving the expression of guide polynucleotide is selected from the group consisting of U6 promoter, tRNAVal promoter, and H1 promoter, and wherein the promoter driving the expression of dCas13a protein is selected from the group consisting of chicken β-actin promoter, SV40 promoter, CMV promoter, Ubc promoter, EF-1α, and CAG promoter.
19. A composition comprising a recombinant expression vector, said recombinant expression vector comprising:
(a) a guide polynucleotide complementary to a target sequence, operably linked to a promoter, wherein the target sequence has a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and wherein the guide polynucleotide has contiguous nucleotides complementary to the target sequence in the range of 24-30 nucleotides, and wherein the guide polynucleotide is complementary to a region within at least 50 nucleotides downstream of a canonical stop codon or pre-mature stop codon of the target sequence; and
(b) a polynucleotide encoding dCas13 protein having a nucleotide sequence selected from SEQ ID NO: 5, and SEQ ID NO: 31, operably linked to a promoter.
20. The composition as claimed in claim 19, wherein the guide polynucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 30.
21. The composition as claimed in claim 19, wherein the promoter driving the expression of the guide polynucleotide is selected from the group consisting of U6 promoter, tRNAVal promoter, and H1 promoter, and wherein the promoter driving the expression of dCas13 protein is selected from the group consisting of chicken β-actin promoter, SV40 promoter, CMV promoter, Ubc promoter, EF-1α, and CAG promoter.
22. The composition as claimed in claim 19, wherein the composition further comprises excipients.
23. The composition as claimed in claim 19, wherein the composition induces translational readthrough across the canonical stop codon or the pre-mature stop codon of the target sequence.
24. The composition as claimed in claim 19, wherein the composition is delivered to a subject for treating a disease.
25. The composition as claimed in claim 24, wherein the composition is delivered by a method selected from the group consisting of Lentivirus, Adeno associated virus (AAV) systems and lipid based nano carriers.
26. The composition as claimed in claim 24, wherein the disease is selected from the group consisting of β-thalassemia, duchenne muscular dystrophy, cystic fibrosis, hemophilia, cancer, retinopathies, usher syndrome, hurler syndrome, spinal muscular atrophy, cystinosis, and infantile neuronal ceroid lipofuscinosis.
27. A guide polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 30.
28. The method as claimed in anyone of the claims 7-11, wherein the guide RNA is capable of hybridizing to the target sequence in transcript-selective and selective manner.