Engineering of systems, methods and optimized guide compositions for sequence manipulation

• US 8,906,616 B2
• Filed: 05/29/2014
• Issued: 12/09/2014
• Est. Priority Date: 12/12/2012
• Status: Active Grant

• 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, Hsu, Patrick, Ran, Fei
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US14/290,575
• Publication Number:
  US 20140273232A1
• Time in Patent Office: 194 Days
• Field of Search: 435/6.1, 435/440, 536/22.1
• US Class Current: 435/6.1
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• 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 15/52 Genes encoding for enzymes or proenzymes
• 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 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/902 using homologous recombination
• 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 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 2800/10 Plasmid DNA
• CPC Class Codes: C12N 2810/50 Vectors comprising as targeting moiety peptide derived from defined protein
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Y 301/00 : Hydrolases acting on ester bonds (3.1)
• 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. An engineered, non-naturally occurring composition comprising a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system having a guide RNA polynucleotide sequence, wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,

(b) a tracr mate sequence, and

(c) a tracr sequence

wherein (a), (b) and (c) are arranged in a 5′

to 3′

orientation,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,

wherein the to CRISPR complex comprises a Type II Cas9 protein complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,

wherein in the polynucleotide sequence, one or more of the guide, tracr and tracr mate sequences are modified.

2. The composition of claim 1, wherein the modified guide RNA comprises a chimeric guide sequence and a tracr sequence.

3. The composition of claim 1, wherein the modification comprises an engineered secondary structure.

4. The composition of claim 1, wherein the modification comprises a reduction in a region of hybridization between the tracr mate sequence and the tracr sequence from the wild type.

5. The composition of claim 1, wherein the modification comprises fusing the tracr mate sequence and the tracr sequence through an artificial loop.

6. The composition of claim 1, wherein the modification comprises the tracr sequence having a length between 40 and 120 nucleotides.

7. The composition of claim 1, wherein the tracr sequence comprises at least nucleotides 1-67 of the corresponding wild type S. pyogenes Cas9 tracRNA.

8. The composition of claim 1, wherein the tracr sequence comprises at least nucleotides 1-85 of the corresponding wild type S. pyogenes Cas9 tracRNA.

9. The composition of claim 1, wherein the modification comprises sequence optimization.

10. The composition of claim 9, wherein the modification comprises adding a polyT terminator sequence in the guide sequence, tracr and/or tracr mate sequence(s).

11. The composition of claim 9 wherein the modification comprises a reduction from the wild type in the number of T nucleotides in polyT regions of the guide sequence tracr and/or tracr mate sequence(s).

12. The composition of claim 10, wherein one or more Ts present in a poly-T sequence of the corresponding wild type sequence have been substituted with a non-T nucleotide.

13. The composition of claim 12, wherein the modified sequence does not comprise any polyT sequence having more than 4 contiguous Ts.

14. The composition of claim 1, wherein the modification comprises altering loops and/or hairpins.

15. The composition of claim 14, wherein the modification comprises providing a minimum of two hairpins.

16. The composition of claim 14, wherein the modification comprises providing at most five hairpins.

17. The composition of claim 14, wherein the modification comprises providing a hairpin formed by complementation between the tracr and tracr mate sequence.

18. The composition of claim 15, wherein the modification comprises providing one or more further hairpin(s) at the 3′

end of the tracrRNA sequence.

19. The composition of claim 15, wherein the modification comprises providing one or more additional hairpin(s) added to the 3′

of the guide sequence.

20. The composition of claim 15, wherein the modification comprises extending the 5′

end of the guide sequence.

21. The composition of claim 20, wherein the modification comprises providing one or more hairpins in the 5′

end of the guide sequence.

22. The composition of claim 20, wherein the modification comprises appending the sequence (5′

-AGGACGAAGTCCTAA) (SEQ ID NO;

1) to the 5′

end of the guide sequence.

23. The composition of claim 1, wherein the modification comprises providing cross linking or providing one or more modified nucleotides in the polynucleotide sequence.

24. The composition of claim 23, wherein modified nucleotides are provided in any or all of the tracr, tracr mate, and/or guide sequences, and/or in a Cas enzyme coding sequence when the CRISPR-Cas system is expressed from coding sequence(s), and/or in vector sequences when the CRISPR-Cas system is expressed from coding sequence(s).

25. The composition of claim 23, wherein providing modified nucleotides comprises inclusion of at least one non naturally occurring nucleotide, or a modified nucleotide, or analogs thereof.

26. The composition of claim 25, wherein the modified nucleotides are modified at the ribose, phosphate, and/or base moiety.

27. The composition of claim 25, wherein the modified nucleotide is selected from the group consisting of 2′

-O-methyl analogs, 2′

-deoxy analogs, or 2′

-fluoro analogs.

28. The composition of claim 25, wherein the modified nucleotide is selected from the group consisting of 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine.

29. A method for genome engineering, the method comprising introducing the composition of claim 1 into a eukaryotic cell containing and expressing a DNA molecule having the target sequence and encoding at least one gene product.

30. The method of claim 29, wherein the genome engineering comprises modifying the DNA molecule by inserting a polynucleotide, said insertion resulting in an insertion, deletion, or substitution of one or more nucleotides.