RNA-guided human genome engineering

• US 9,970,024 B2
• Filed: 12/16/2013
• Issued: 05/15/2018
• 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, Yang, Luhan
• Primary Examiner(s): Nguyen, Quang
• Application Number:
  US14/653,144
• Publication Number:
  US 20160032274A1
• Time in Patent Office: 1,611 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. A method of visualizing a target nucleic acid sequence in a eukaryotic cell comprising providing to the eukaryotic cell a guide RNA complementary to the target nucleic acid sequence, and providing to the cell a Cas 9 protein having inactive nuclease domains and having a fluorescent molecule attached thereto, wherein the guide RNA and the Cas 9 protein having the fluorescent molecule attached thereto form a complex at the target nucleic acid sequence and is visualized.

2. The method of claim 1 wherein the fluorescent molecule is a fluorescent protein, a protein bound organic fluorophore, a nucleic acid bound organic fluorophore, a quantum dot, a molecular beacon, an echo probe or a molecular beacon replacement.

3. The method of claim 1 wherein the eukaryotic cell is a yeast cell, a plant cell, a mammalian cell, a stem cell or a human cell.

4. The method of claim 1 wherein the RNA includes between about 10 to about 250 nucleotides.

5. The method of claim 1 wherein the RNA includes between about 20 to about 100 nucleotides.

6. The method of claim 1 wherein the eukaryotic cell is provided with a plurality of guide RNAs complementary to different target nucleic acids, wherein the guide RNAs and the Cas 9 protein having the fluorescent molecule attached thereto form complexes at the target nucleic acid sequences and are visualized.

7. The method of claim 1 wherein the guide RNA is provided to the eukaryotic cell by introducing to the eukaryotic cell a nucleic acid encoding the guide RNA, wherein the Cas 9 protein having the fluorescent molecule attached thereto is provided to the eukaryotic cell by introducing to the eukaryotic cell a nucleic acid encoding the Cas 9 protein having the fluorescent molecule attached thereto, and wherein the eukaryotic cell expresses the guide RNA and the Cas 9 protein having the fluorescent molecule attached thereto.