COMPOSITIONS AND METHODS DIRECTED TO CRISPR/CAS GENOMIC ENGINEERING SYSTEMS

• US 20140273037A1
• Filed: 03/14/2014
• 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 9,234,213 B2
• Time in Patent Office: Days
• US Class Current: 435/8
• 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.

8. An expression construct comprising:

(a) a first polynucleotide encoding a prokaryote Cas9 protein, and

(b) a second polynucleotide encoding a guide RNA, said guide RNA comprising (i) a crRNA-tracrRNA chimeric transcript, and (ii) a targeting sequence corresponding to a genomic target of interest, wherein the targeting sequence is operably linked to the crRNA-tracrRNA chimeric transcript.

9. The expression construct of claim 8, wherein the expression construct is a mammalian expression construct.

10. The expression construct of claim 8, wherein the prokaryote Cas9 protein is a Streptococcus pyogenes Cas9 protein.

11. The expression construct of claim 8, wherein the first polynucleotide is optimized for human codon utilization.

12. The expression construct of claim 8, wherein the first polynucleotide comprises the nucleotide sequence of positions 91 through 4191 of SEQ ID NO:

1.

13. The expression construct of claim 12, wherein the first polynucleotide further encodes at least one nuclear localization signal.

14. The expression construct of claim 8, wherein the first polynucleotide comprises the nucleotide sequence of positions 40 through 4242 of SEQ ID NO:

1.

15. The expression construct of claim 8, wherein the first polynucleotide is a native prokaryotic polynucleotide, or derived from a native prokaryotic polynucleotide.

16. A system for targeted genomic modification in mammalian cells, the system comprising:

(a) a mammalian expression construct comprising;

i) a first polynucleotide encoding a prokaryotic Cas9 protein, and

ii) a second polynucleotide encoding a guide RNA, said guide RNA comprising;

(A) a crRNA-tracrRNA chimeric transcript, and (B) a targeting sequence operably linked to the crRNA-tracrRNA chimeric transcript, 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 after delivery into the mammalian host cell.

17. The system of claim 16, 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.

18. The system of claim 16, wherein the system is a multiplex system, further comprising at least a second guide RNA comprising a crRNA-tracrRNA chimeric transcript that is operably linked to a second targeting sequence corresponding to a second genomic target of interest.

19. A method for targeted genomic modification in mammalian cells, the system comprising:

(a) providing;

i) a mammalian expression construct, comprising;

A) a first polynucleotide encoding a prokaryotic Cas9 protein, and B) a second polynucleotide encoding a guide RNA, said guide RNA comprising;

(I) a crRNA-tracrRNA chimeric transcript, and

(II) a targeting sequence operatively linked to the crRNA-tracrRNA chimeric transcript, said targeting sequence corresponding to a genomic target of interest,

ii) a mammalian host cell comprising a genomic target of interest corresponding to said targeting sequence, and

iii) a means for delivering said mammalian expression construct into said mammalian host cell; 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.

20. The method of claim 19, further comprising:

(c) identifying or selecting for host cells having a genomic modification that is induced by the mammalian expression construct.