Crispr/cas systems for genomic modification and gene modulation
AssigneeSystem Biosciences, LLC
InventorFangting Wu
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Abstract
The invention relates to engineered CRISPR/Cas9 systems for genomic modification and regulation of gene expression in mammalian cells. The specification describes the design and validation of polynucleotides encoding the Streptococcus pyogenes (S. pyogenes) Cas9 gene and protein and variants of that protein, where the nucleotide sequence has been optimized for expression in mammalian cells, and also modified by fused sequences that enhance various aspects of the CRISPR/Cas system. The specification also describes systems for RNA-guided genome engineering and gene regulation in mammalian cells, including human cells.
Claims27
A polynucleotide comprising a nucleotide sequence selected from SEQ ID NOs:
- 3, 4 and 6-10, conservative variants thereof, and subsequences thereof which are at least 100 nucleotides in length.
A polypeptide comprising an amino acid sequence selected from SEQ ID NOs:
- 5, 11, 12 and 13, and conservative variants thereof.
An expression construct comprising:
- (a) a first polynucleotide encoding a bacterial Cas9 protein or a variant thereof or a fusion protein therewith, and
- (b) a second polynucleotide encoding a guide RNA, said guide RNA comprising (i) a crRNA-tracrRNA scaffold polynucleotide, and (ii) a targeting sequence corresponding to a genomic target of interest, wherein the targeting sequence is operably linked to the crRNA-tracrRNA scaffold polynucleotide.
A method for targeted genomic modification in mammalian cells, the method comprising:
- (a) providing:
- i) a mammalian expression construct comprising:
- A) a first polynucleotide encoding a bacterial Cas9 protein, or a variant thereof or a fusion protein therewith, and
- B) a second polynucleotide encoding a guide RNA, said guide RNA comprising:
- (I) a crRNA-tracrRNA scaffold polynucleotide, and
- (II) a targeting sequence operably linked to the crRNA-tracrRNA scaffold polynucleotide, said targeting sequence corresponding to a genomic locus of interest,
- ii) a mammalian host cell comprising the genomic locus of interest, and
- (b) delivering said mammalian expression construct into said mammalian host cell wherein the first and second polynucleotides are expressed within the mammalian host cell.
A method for targeted genomic modification in mammalian cells, the method comprising:
- (a) providing:
- i) a first polynucleotide comprising the nucleotide sequence of either SEQ ID NO:
- 3, 7 or conservative variants thereof,
- ii) a second polynucleotide encoding a guide RNA, said guide RNA comprising:
- A) a crRNA-tracrRNA scaffold polynucleotide, and
- B) a targeting sequence operably linked to the crRNA-tracrRNA scaffold polynucleotide, said targeting sequence corresponding to a genomic locus of interest,
- iii) a mammalian host cell comprising the genomic locus of interest, and
- (b) delivering said first and second polynucleotides into said mammalian host cell.
A system for targeted genomic modification in mammalian cells, the system comprising:
- (a) a mammalian expression construct comprising:
- i) a first polynucleotide encoding a bacterial Cas9 protein, or a variant thereof or a fusion protein therewith, and
- ii) a second polynucleotide encoding a guide RNA, said guide RNA comprising:
- (A) a crRNA-tracrRNA scaffold polynucleotide, and
- (B) a targeting sequence operably linked to the crRNA-tracrRNA scaffold polynucleotide, said targeting sequence corresponding to a genomic target of interest,
- (b) a mammalian host cell comprising the genomic target of interest, and
- (c) a means for delivering said mammalian expression construct into said mammalian host cell, wherein the first and second polynucleotides are expressed inside the mammalian host cell after delivery of the mammalian expression construct into the mammalian host cell.
A method for regulating the expression of a gene of interest in a mammalian host cell, the method comprising:
- (a) providing:
- i) a mammalian expression construct comprising:
- A) a first polynucleotide encoding a fusion polypeptide comprising (i) a variant bacterial Cas9 protein that is a null nuclease, and (ii) a transcriptional regulatory domain operably fused to the Cas9 protein, and
- B) a second polynucleotide encoding a guide RNA, said guide RNA comprising:
- (I) a crRNA-tracrRNA scaffold polynucleotide, and
- (II) a targeting sequence operably linked to the crRNA-tracrRNA scaffold polynucleotide, said targeting sequence corresponding to a genomic locus that is in proximity to the gene of interest,
- ii) a mammalian host cell comprising the gene of interest, and
- (b) delivering said mammalian expression construct into said mammalian host cell wherein the first and second polynucleotides are expressed within the mammalian host cell.
A method for regulating the expression of a gene of interest in a mammalian host cell, the method comprising:
- (a) providing:
- i) a first polynucleotide comprising the nucleotide sequence of SEQ ID NO:
- 9 or a conservative variant thereof, encoding a fusion polypeptide comprising (i) a variant Cas9 protein that is a null nuclease, and (ii) a transcriptional regulatory domain operably fused to the Cas9 protein, and
- ii) a second polynucleotide encoding a guide RNA, said guide RNA comprising:
- A) a crRNA-tracrRNA scaffold polynucleotide, and
- B) a targeting sequence operably linked to the crRNA-tracrRNA scaffold polynucleotide, said targeting sequence corresponding to a genomic locus that is in proximity to the gene of interest,
- iii) a mammalian host cell comprising the gene of interest, and
- (b) delivering said first and second polynucleotides into said mammalian host cell wherein the first and second polynucleotides are expressed within the mammalian host cell.
Dependencies are inferred from the claim text.