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

• US 20200032277A1
• Filed: 06/18/2019
• Published: 01/30/2020
• Est. Priority Date: 12/12/2012
• Status: Abandoned 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:
  US16/445,150
• Publication Number:
  US 20200032277A1
• 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 of modifying a target DNA molecule, the method comprising:

allowing a CRISPR complex to target a target sequence of the target DNA molecule, said CRISPR complex comprising;

(a) a Cas9; and

(b) RNA comprising;

(i) a guide sequence linked to a tracr mate sequence, wherein the guide sequence is capable of hybridizing to the target sequence, and

(ii) a tracr sequence that hybridizes to the tracr mate sequence to form a duplex structure,

wherein said allowing a CRISPR complex to target a target sequence of the target DNA molecule takes place in vitro or in a human or non-human animal or plant, or in a cell or tissue thereof,

resulting in modification of the target DNA molecule.

2. The method of claim 1, wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two.

3. The method of claim 1, wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two, wherein the shorter of the two has a length comprising about 12 to about 27 nucleotides.

4. The method of claim 1, wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two, wherein the shorter of the two has a length comprising about 15 to about 27 nucleotides.

5. The method of claim 1, wherein the duplex structure has a total of 10, 11, 17, or 21 complementary pairs.

6. The method of claim 1, wherein the duplex structure has a total of 11 or 17 complementary pairs.

11. The method of claim 1 wherein said modification of the target DNA molecule is cleavage of the target DNA molecule.

12. The method of claim 1, wherein the target sequence is at least 15 nucleotides (nt) to 20 nt long.

13. The method of claim 1, wherein the target sequence is at least 15 nucleotides (nt) to 25 nt long.

14. The method of claim 1, wherein the target DNA is chromosomal DNA.

15. The method of claim 1, wherein the tracr sequence comprises UAGCAAGUUAAAAUAAGGCUAGUCCGUUUU.

16. The method of claim 1, wherein the RNA comprises one or more modified nucleotides.

17. The method of claim 1, wherein the guide sequence and/or the tracr sequence is conjugated to an avidin/biotin complex, an acridine, fluorescent label or fluorcoumarin.

18. The method of claim 1, wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two when optimally aligned.

7. A method of modifying a target DNA molecule, the method comprising:

allowing a CRISPR complex to target a target sequence of the target DNA molecule, said CRISPR complex comprising;

(a) a Cas9; and

(b) RNA comprising;

(i) a guide sequence linked to a tracr mate sequence, wherein the guide sequence is capable of hybridizing to the target sequence, and

(ii) a tracr sequence that hybridizes to the tracr mate sequence to form a duplex structure,

wherein the duplex structure has a total number of Watson-Crick base pairs in the range of 9-19,

wherein said allowing a CRISPR complex to target a target sequence of the target DNA molecule takes place in vitro or in a human or non-human animal or plant, or in a cell or tissue thereof,

resulting in modification of the target DNA molecule.

8. The method of claim 7, wherein the duplex structure has a total number of Watson-Crick base pairs in the range of 9-16.

9. The method of claim 7, wherein the duplex structure has a total of 9, 16, or 19 Watson-Crick base pairs.

10. The method of claim 7, wherein the duplex structure has a total of 9 or 16 Watson-Crick base pairs.

19. A method for cleaving or editing a target DNA molecule having a target sequence or modulating transcription of at least one gene target having the target sequence with an engineered Type II CRISPR complex, the method comprising allowing the CRISPR complex to target the target DNA molecule or the gene target,

wherein the CRISPR complex comprises:

a) RNA comprising (i) an engineered guide sequence linked to a tracr mate sequence wherein the guide sequence is capable of hybridizing with the target sequence and (ii) a tracr sequence that hybridizes to the tracr mate sequence to form a duplex structure, and

b) a Cas9,

wherein the RNA forms the complex with the Cas9 and targets the Cas9 of the complex to the target DNA molecule or the gene target, whereby said target DNA molecule is cleaved or edited or transcription of the gene target is modulated.

20. The method of claim 19, wherein each of (i) and (ii) are separate molecules and wherein at least a portion of the tracr mate sequence in (i) hybridizes to at least a portion of (ii).

21. The method of claim 19, wherein the tracr mate sequence in (i) and the tracr sequence in (ii) hybridizes to form a duplex structure.

22. The method of claim 21, wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two.

23. The method of claim 21, wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two, wherein the shorter of the two has a length comprising about 12 to about 27 nucleotides.

24. The method of claim 21, wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two, wherein the shorter of the two has a length comprising about 15 to about 27 nucleotides.

25. The method of claim 21, wherein the duplex structure has a total number of Watson-Crick base pairs in the range of 9-19.

26. The method of claim 21, wherein the duplex structure has a total number of Watson-Crick base pairs in the range of 9-16.

27. The method of claim 21, wherein the duplex structure has a total of 9, 16, or 19 Watson-Crick base pairs.

28. The method of claim 21, wherein the duplex structure has a total of 10, 11, 17, or 21 complementary pairs.