CRISPR/CAS SYSTEMS FOR GENOMIC MODIFICATION AND GENE MODULATION

• US 20140273226A1
• Filed: 03/17/2014
• Published: 09/18/2014
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: System Biosciences, LLC
• Original Assignee: System Biosciences, LLC
• Inventors: Wu, Fangting
• Granted Patent: US 9,738,908 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• 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 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.

Claims

1. 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.

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

3, 7 or 9, and wherein the polynucleotide further comprises at least one operatively linked nucleotide sequence encoding a nuclear localization signal.

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

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

5. An expression construct comprising the polynucleotide of claim 1, where the expression construct is a human expression construct.

6. An mRNA expression construct comprising:

(i) a polynucleotide of claim 1, and (ii) an in vitro transcription promoter element that is operably linked to the polynucleotide of claim 1.

7. An mRNA expression construct comprising:

(i) a polynucleotide of claim 1, and (ii) a bacteriophage promoter element that is operably linked to the polynucleotide of claim 1.

8. A polypeptide comprising an amino acid sequence selected from SEQ ID NOs:

5, 11, 12 and 13, and conservative variants thereof.

9. A polynucleotide encoding a polypeptide of claim 8.

10. 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.

11. The expression construct of claim 10, wherein the expression construct is a mammalian expression construct.

12. The expression construct of claim 10, wherein the bacterial Cas9 protein is the wild type Streptococcus pyogenes Cas9 protein.

13. The expression construct of claim 10, wherein the first polynucleotide is optimized for mammalian codon utilization.

14. The expression construct of claim 10, wherein the first polynucleotide comprises nucleotide sequence encoding at least one nuclear localization signal.

15. 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.

16. The method of claim 15, further comprising:

(c) visualizing, identifying or selecting for host cells having a genomic modification at the genomic locus of interest that is induced by the delivering the mammalian expression construct into the mammalian host cell.

17. 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.

18. The method of claim 17, further providing a donor polynucleotide comprising nucleotide sequence having homology to the genomic target of interest, and delivering said donor polynucleotide into said mammalian host cell concurrently with said first and second polynucleotides.

19. The method of claim 17, wherein said first and second polynucleotides are RNA polynucleotides.

20. The method of claim 17, wherein the method is a method for multiplexed genomic modification, further comprising at least a second guide RNA comprising a crRNA-tracrRNA scaffold polynucleotide that is operably linked to at least a second targeting sequence corresponding to a second genomic target of interest.

21. The method of claim 17, further comprising:

(c) visualizing, identifying or selecting for host cells having a genomic modification at the genomic locus of interest.

22. 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.

23. The system of claim 22, further comprising a donor polynucleotide comprising nucleotide sequence having homology to the genomic target of interest, wherein the donor polynucleotide and the genomic target of interest undergo homologous recombination within the mammalian host cell, thereby producing a targeted genomic modification.

24. The system of claim 22, wherein the mammalian expression construct is a human expression construct and the mammalian host cell is a human host cell.

25. The system of claim 22, wherein the system is a multiplex system, further comprising at least a second guide RNA comprising a crRNA-tracrRNA scaffold polynucleotide that is operably linked to a second targeting sequence corresponding to a second genomic target of interest.

26. 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.

27. 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.