Compositions and methods directed to CRISPR/Cas genomic engineering systems
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.
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 .