CRISPR-BASED GENOME MODIFICATION AND REGULATION

• US 20180135073A1
• Filed: 07/18/2017
• Published: 05/17/2018
• Est. Priority Date: 03/15/2013
• Status: Abandoned Application

• Current Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Original Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Inventors: Chen, Fuqiang, Davis, Gregory D.
• Application Number:
  US15/653,136
• Publication Number:
  US 20180135073A1
• Time in Patent Office: Days
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention provides methods for modifying chromosomal sequences. In particular, methods are provided for using RNA-guided endonucleases or modified RNA-guided endonucleases to modify targeted chromosomal sequences.

Claims

1. A method for modifying a chromosomal sequence in a cell or embryo, the method comprising introducing into the cell or embryo (a) two or more RNA-guided endonucleases or nucleic acid encoding two or more RNA-guided endonucleases and (b) two or more guiding RNAs or DNA encoding two or more guiding RNAs, wherein each guiding RNA guides one of the RNA-guided endonucleases to a targeted site in the chromosomal sequence and the RNA-guided endonuclease cleaves at least one strand of the chromosomal sequence at the targeted site.

2. The method of claim 1, wherein each RNA-guided endonuclease is derived from a Cas9 protein and comprises at least two nuclease domains.

3. The method of claim 2, wherein one of the nuclease domains of each of the two RNA-guided endonucleases is modified such that each RNA-guided endonuclease cleaves one strand of a double-stranded sequence, and wherein the two RNA-guided endonucleases together introduce a double-stranded break in the chromosomal sequence that is repaired by a DNA repair process such that the chromosomal sequence is modified.

4. The method of claim 1, wherein each RNA-guided endonuclease introduces a double-stranded break in the chromosomal sequence that is repaired by a DNA repair process such that the chromosomal sequence is modified.

5. The method of claim 1, further comprising introducing into the cell at least one donor polynucleotide, wherein the donor polynucleotide comprises at least one sequence having substantial sequence identity with sequence on one side of the targeted site in the chromosomal sequence.

6. The method of claim 5, wherein the donor polynucleotide further comprises a donor sequence.

7. The method of claim 6, wherein the donor sequence is integrated into or exchanged with the chromosomal sequence.

8. The method of claim 1, wherein the cell is a human cell, a non-human mammalian cell, a stem cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.

9. The method of claim 1, wherein the embryo is a non-human one cell embryo.

10. The method of claim 1, wherein each RNA-guided endonuclease further comprises at least one additional domain chosen from a nuclear localization signal, a cell-penetrating domain, or a marker domain.

11. The method of claim 1, wherein each RNA-guided endonuclease is derived from a Cas9 protein and comprises one functional nuclease domain.

12. The method of claim 11, wherein each RNA-guided endonuclease further comprises at least one nuclear localization signal.