Compositions and methods directed to CRISPR/Cas genomic engineering systems

• US 9,234,213 B2
• Filed: 03/14/2014
• Issued: 01/12/2016
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: System Biosciences, LLC
• Original Assignee: System Biosciences, LLC
• Inventors: Wu, Fangting
• Primary Examiner(s): Joike, Michele K
• Assistant Examiner(s): Hammell, Neil P
• Application Number:
  US14/211,858
• Publication Number:
  US 20140273037A1
• Time in Patent Office: 669 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2800/22 Vectors comprising a coding region that has been codon optimised for expression in a respective host
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The invention relates to engineered CRISPR/Cas9 systems for genomic modification in mammalian cells. The present specification describes the design and testing of a polynucleotide encoding the Streptococcus pyogenes (S. pyogenes) Cas9 protein, where the nucleotide sequence has been optimized for expression in mammalian cells. The specification also describes all-in-one systems for RNA-guided genome engineering in mammalian cells, including human cells.

Claims

1. A polynucleotide comprising the nucleotide sequence of positions 91 through 4191 of SEQ ID NO:

1.

2. The polynucleotide of claim 1, wherein the polynucleotide comprises the nucleotide sequence of positions 40 through 4242 of SEQ ID NO:

1.

3. The polynucleotide of claim 1, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:

1.

4. The polynucleotide of claim 1, wherein the polynucleotide further comprises at least one operably linked nucleotide sequence encoding a nuclear localization signal.

5. An expression construct comprising the polynucleotide of claim 1.

6. An expression construct comprising the polynucleotide of claim 1, where the expression construct is a mammalian expression construct.

7. An mRNA expression construct comprising:

(i) the polynucleotide of claim 1, and (ii) a second polynucleotide comprising a T7 promoter, wherein the T7 promoter is operably linked to the polynucleotide of claim 1.