CRISPR-CAS COMPONENT SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION

• US 20190017058A1
• Filed: 04/30/2018
• Published: 01/17/2019
• Est. Priority Date: 12/12/2012
• Status: Active Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Zhang, Feng, CONG, Le, COX, David Benjamin Turitz, HSU, Patrick, LIN, Shuailiang, RAN, Fei, PLATT, Randall Jeffrey, SANJANA, Neville Espi
• Application Number:
  US15/967,495
• Publication Number:
  US 20190017058A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/10 Type of nucleic acid
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/101 for bacteria
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: G16B 20/00 : ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 : Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/30 : Detection of binding sites or motifs
• CPC Class Codes: G16B 20/50 : Mutagenesis
• CPC Class Codes: G16B 30/00 : ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 : Sequence alignment; Homology search

Abstract

The invention provides for systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/CAS system.

Claims

1. A method for cleaving, editing, or modifying at least one sequence adjacent to a Protospacer Adjacent Motif (PAM) in a eukaryotic cell, comprising:

delivering a non-naturally occurring or engineered single eukaryotic expression vector to the eukaryotic cell,

wherein the expression vector comprises;

a) at least one nucleotide sequence encoding at least one chimeric construct comprising a tracr sequence comprising 30 or more nucleotides in length, and

b) at least one nucleotide sequence encoding at least one Cas9,

wherein;

components a) and b) are each operably linked to a regulatory element for transcription thereof in the eukaryotic cell and the expression vector includes at least one regulatory element therefor,

the chimeric construct is designed to form with Cas9 a complex,

the chimeric construct comprise(s) guide(s) designed to hybridize with the target sequence(s),

the chimeric construct and Cas9 do not naturally occur together, and

the complex comprising the chimeric construct and Cas9 does not naturally occur, whereby there is cleaving, editing, or modifying of the target sequence.

2. The method of claim 1, wherein the method comprises a multiplexed method for cleaving, editing, or modifying more than one target sequence.

3. The method of claim 1, wherein at least one gene target comprises the at least one target sequence and the method is for modulating transcription or altering expression of the gene target.

4. The method of claim 1, wherein the regulatory element comprises a polymerase III promoter.

5. The method of claim 1, wherein the regulatory element comprises a polymerase II promoter.

6. The method of claim 1, wherein the chimeric construct includes a tracr mate sequence, the tracr sequence comprises 30-50 nucleotides or more than 50 nucleotides, and exhibits at least 50% sequence complementarity along the length of the tracr mate.

7. The method of claim 1, wherein component b) is codon-optimized for expression in the eukaryotic cell.

8. The method of claim 1, wherein the guide includes a guide sequence comprising at least 15 nucleotides.

9. The method of claim 1, wherein the chimeric construct comprises two, three, four or five hairpins.

10. The method of claim 1, wherein Cas9 includes at least one nuclear localization signal (NLS).

11. The method of claim 10, wherein Cas9 includes at least one NLS at or in the proximity of the C-terminus of Cas9, or at least one NLS at or in the proximity of the N-terminus of Cas9, or at least one NLS at or in the proximity of the C-terminus of Cas9 and at least one NLS at or in the proximity of the N-terminus of Cas9.

12. The method of claim 1, wherein the method is for cleaving the target sequence.

13. The method of claim 1, wherein the method is for editing the target sequence.

14. The method of claim 1, wherein the method is for modifying the target sequence.

15. The method of claim 3, wherein the method is for modulating transcription of the gene target.

16. The method of claim 3, wherein the method is for altering expression of the gene target.

17. The method of claim 1, wherein the expression vector comprises a lentivirus, an adenovirus or an adeno-associated virus.

18. The method of claim 1, wherein the eukaryotic cell comprises a plant cell.

19. The method of claim 1, wherein the eukaryotic cell comprises a mammalian cell.

20. The method of claim 1, wherein Cas9 includes one or more effector domains comprising a transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, transcription-protein recruiting domain, cellular uptake activity associated domain, nucleic acid binding domain or antibody presentation domain.

21. The method of claim 1, wherein there is at least one first regulatory element for transcription of component a) and at least one second regulatory element for transcription of component b).

22. The method of claim 2, wherein the multiplexed method comprises the chimeric construct designed to hybridize with more than one target sequence comprising component a) comprising coding for more than one chimeric construct.

23. The method of claim 22, wherein there is more than one regulatory element for transcription of the more than one chimeric construct of component a).

24. The method of claim 20, wherein the one or more effector domains comprises a nuclease domain.

25. The method of claim 19, wherein the method includes isolating the mammalian cell from a subject prior to the delivering.

26. The method of claim 1, wherein two strands of DNA of the target sequence are cleaved.

27. The method of claim 1, including delivering a donor polynucleotide.

28. The method of claim 1, wherein the chimeric construct comprises one or more modified nucleotides, nucleotide analogs or non-nucleotide components.

29. The method of claim 19, wherein the mammalian cell is a human cell.

30. A non-naturally occurring or engineered single eukaryotic expression vector comprising:

a) at least one nucleotide sequence encoding at least one chimeric construct comprising a tracr sequence comprising 30 or more nucleotides in length, and

b) at least one nucleotide sequence encoding at least one Cas9,

wherein;

components a) and b) are each operably linked to a regulatory element for transcription thereof in a eukaryotic cell and the expression vector includes at least one regulatory element therefor,

the chimeric construct is designed to form with Cas9 a complex,

the chimeric construct comprise(s) guide(s) designed to hybridize with target sequence(s) in the eukaryotic cell,

the chimeric construct and Cas9 do not naturally occur together, and

the complex comprising the chimeric construct and Cas9 does not naturally occur.

31-57. -57. (canceled)