RNA-Guided Human Genome Engineering

• US 20190249194A1
• Filed: 04/29/2019
• Published: 08/15/2019
• Est. Priority Date: 12/17/2012
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M., Mali, Prashant G., Yang, Luhan
• Granted Patent: US 11,359,211 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1024 In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 2810/55 from bacteria
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

Abstract

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

Claims

1-14. -14. (canceled)

15. A method of altering a eukaryotic cell comprising

providing the eukaryotic cell with a guide RNA having a spacer sequence complementary to genomic DNA of the eukaryotic cell and a scaffold sequence,

providing the eukaryotic cell with a codon optimized Cas9 of a Type II CRISPR system that interacts with the guide RNA and cleaves the genomic DNA in a site specific manner,

wherein the guide RNA binds to complementary genomic DNA and the Cas enzyme of a Type II CRISPR system cleaves the genomic DNA in a site specific manner, and wherein the codon optimized Cas9 is a human codon optimized Cas9 encoded by a nucleic acid comprising SEQ ID NO:

22.

16. The method of claim 15 wherein the eukaryotic cell is provided with the guide RNA by introducing into the eukaryotic cell a nucleic acid encoding the guide RNA and wherein the eukaryotic cell is provided with the Cas enzyme of a Type II CRISPR system by introducing into the eukaryotic cell a nucleic acid encoding the Cas enzyme of a Type II CRISPR system.

17. The method of claim 15 wherein the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.

18. The method of claim 15 wherein the eukaryotic cell is a human cell.

19. The method of claim 15 wherein the eukaryotic cell is a stem cell.

20. The method of claim 15 wherein the eukaryotic cell is human induced pluripotent stem cell.

21. The method of claim 15 comprising altering the eukaryotic cell at a plurality of genomic DNA sites comprising

providing the eukaryotic cell with a plurality of guide RNAs, each guide RNA having a spacer sequence complementary to a different site on genomic DNA of the eukaryotic cell and a spacer sequence,

wherein the guide RNAs bind to complementary genomic DNA and the Cas enzyme of a Type II CRISPR system cleaves the genomic DNA in a site specific manner.

22. The method of claim 25 wherein the eukaryotic cell is provided with the plurality of guide RNAs by introducing into the eukaryotic cell a plurality of nucleic acids encoding the guide RNAs.

23. The method of claim 15 wherein the guide RNA is a crRNA-tracrRNA fusion transcript.

24. The method of claim 15 wherein the guide RNA is a crRNA-tracrRNA fusion transcript having a secondary structure comprising a first hairpin connected to the spacer sequence and a second 3′

hairpin.

25. The method of claim 15 wherein the guide RNA is expressed by the eukaryotic cell using a human U6 polymerase III promoter.

26. The method of claim 15 wherein the guide RNA includes 100 nucleotides.

27. The method of claim 15 wherein the Cas enzyme cleaves the target nucleic acid sequence and a nucleotide is deleted or inserted.

28. The method of claim 15 wherein the Cas enzyme includes a nuclear localization signal.

29. The method of claim 15 wherein the Cas enzyme includes a C-terminus nuclear localization signal.

30. The method of claim 15 wherein the Cas enzyme includes a C-terminus SV40 nuclear localization signal.

31. The method of claim 16 wherein the nucleic acid encoding the guide RNA or the nucleic acid encoding the Cas enzyme of a Type II CRISPR system is introduced into the cell using a viral delivery method.

32. The method of claim 16 wherein the nucleic acid encoding the guide RNA or the nucleic acid encoding the Cas enzyme of a Type II CRISPR system is introduced into the cell using an adeno-associated virus.

33. The method of claim 15 wherein and the Cas enzyme cleaves the target nucleic acid sequence thereby altering expression of the target nucleic acid sequence.

34. The method of claim 15 wherein and the Cas enzyme cleaves the target nucleic acid sequence resulting in nonhomologous end joining.

35. The method of claim 15 further comprising providing the eukaryotic cell with a donor nucleic acid, and

wherein the Cas enzyme cleaves the target nucleic acid sequence, and

wherein the donor nucleic acid sequence is integrated into the target nucleic acid sequence.

36. The method of claim 15 wherein and the Cas enzyme cleaves the target nucleic acid sequence resulting in deletion or insertion.

37. A method of altering a eukaryotic cell comprising

providing the eukaryotic cell with a guide RNA having a spacer sequence complementary to genomic DNA of the eukaryotic cell and a scaffold sequence,

providing the eukaryotic cell with a codon optimized Cas9 of a Type II CRISPR system that interacts with the guide RNA and cleaves the genomic DNA in a site specific manner,

wherein the guide RNA binds to complementary genomic DNA and the Cas enzyme of a Type II CRISPR system cleaves the genomic DNA in a site specific manner,

wherein the guide RNA includes between 100 and 250 nucleotides,

wherein the codon optimized Cas9 is a human codon optimized Cas9 encoded by a nucleic acid comprising SEQ ID NO:

22.

38. An RNA-guided genome editing system comprising

(1) a guide RNA sequence or a first nucleic acid sequence encoding the guide RNA sequence and

(2) a Cas enzyme that forms a complex with the guide RNA sequence, or a second nucleic acid sequence encoding the Cas enzyme,

wherein the guide RNA sequence comprises a spacer sequence complementary to a target nucleic acid sequence and which binds to the target nucleic acid sequence, and wherein the guide RNA sequence is a crRNA-tracrRNA fusion transcript having a secondary structure comprising a first hairpin connected to the spacer sequence and a second 3′

hairpin.

39. The RNA-guided genome editing system of claim 38 wherein the guide RNA includes 100 to 250 nucleotides.

40. The RNA-guided genome editing system of claim 38 wherein the guide RNA includes 100 nucleotides.

41. A eukaryotic cell containing the RNA-guided genome editing system of claim 38.