Granted Patent B2

US 12,188,043 · App. 17/660,523 · Granted Jan 7, 2025

Inventors: Shengkan Jin (Belle Mead, NJ); Juan-Carlos Collantes (Princeton, NJ)

C12N15/902C12N9/22C12N15/102C12N15/11C12N2310/20

Abstract

Claims (42)

1. A system for editing a target DNA or RNA comprising:

(i) a CRISPR protein, or a polynucleotide encoding the same, wherein the CRISPR protein comprises a RuvC-like motif and does not generate a double-stranded break;

(ii) an RNA scaffold, or a DNA polynucleotide encoding the same, said RNA scaffold comprising

(a) a nucleic acid-targeting motif comprising a guide RNA sequence that is complementary to a target nucleic acid sequence,

(b) a CRISPR motif capable of binding to the CRISPR protein, and

(c) a protein-binding RNA motif, and

(iii) a fusion protein, or a polynucleotide encoding the same, said fusion protein comprising

(a) an RNA binding domain capable of binding to the protein-binding RNA motif, and

(b) an effector domain that has an enzymatic activity of changing base-pairing of the target DNA or RNA.

2. The system of claim 1 , wherein the CRISPR protein does not have a nuclease activity.

3. The system of claim 1 , wherein the protein-binding RNA motif and the RNA binding domain are a pair selected from the group consisting of:

a telomerase Ku binding motif and a Ku protein,

a telomerase Ku binding motif and an RNA-binding section of the Ku protein,

a telomerase Sm7 binding motif and an Sm7 protein,

a telomerase Sm7 binding motif and an RNA-binding section of the Sm7 protein,

an MS2 phage operator stem-loop and an MS2 coat protein (MCP),

an MS2 phage operator stem-loop and an RNA-binding section of the MCP,

a PP7 phage operator stem-loop and a PP7 coat protein (PCP),

a PP7 phage operator stem-loop and an RNA-binding section of the PCP,

a SfMu phage Com stem-loop and a Com RNA binding protein,

a SfMu phage Com stem-loop and an RNA-binding section of the Com RNA binding

protein,

a non-natural RNA aptamer and a corresponding aptamer ligand, and

a non-natural RNA aptamer and an RNA-binding section of the aptamer ligand.

4. The system of claim 1 , wherein the RNA binding domain and the effector domain is joined by a linker.

5. The system of claim 4 , wherein the linker comprises 1 to 100 amino acid residues.

6. The system of claim 1 , wherein the enzymatic activity is deamination activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity.

7. The system of claim 6 , wherein the enzymatic activity is deamination activity, methyltransferase activity, or demethylase activity.

8. The system of claim 6 , wherein the enzymatic activity is a cytosine deamination activity or adenosine deamination activity.

9. An isolated nucleic acid encoding components (i)-(iii) of the system of claim 1 .

10. An expression vector comprising the isolated nucleic acid of claim 9 .

11. A host cell comprising the isolated nucleic acid of claim 9 .

12. A method of site-specific sequence alteration of a target nucleic acid, comprising contacting the target nucleic acid with components (i)-(iii) of the system of claim 1 .

13. The method of claim 12 , wherein the target nucleic acid is in a cell.

14. The method of claim 13 , wherein the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell.

15. The method of claim 13 , wherein the cell is isolated from a human or non-human subject.

16. The method of claim 15 , wherein the human or non-human subject has a genetic mutation of a gene.

17. The method of claim 16 , wherein the subject has a disorder caused by the genetic mutation or is at risk of having the disorder.

18. The method of claim 17 , wherein said site-specific sequence alteration corrects the genetic mutation or inactivates the expression of the gene.

19. The method of claim 15 , wherein the subject has a pathogen or is at risk of exposure to the pathogen and said site-specific sequence alteration inactivates a gene of the pathogen.

20. A kit comprising the system of claim 1 .

21. A cell, wherein the cell comprises the system of claim 1 .

Assignments (1)

Continuity (3)

References Cited (92)