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

• US 20200032278A1
• 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:
  US16/445,156
• Publication Number:
  US 20200032278A1
• 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:

contacting a target DNA molecule having a target sequence with a complex comprising;

(a) a Cas9 protein; and

(b) a DNA-targeting RNA comprising;

(i) a targeter-RNA that hybridizes with the target sequence, and

(ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment,

wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 10 to 15 base pairs,

wherein said contacting takes place outside of a bacterial cell and outside of an archaeal cell, thereby resulting in modification of the target DNA molecule.

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

3. The method of claim 1, wherein the target sequence is 15 nucleotides (nt) to 18 nt long.

4. The method of claim 1, wherein the target sequence is 18 nucleotides (nt) to 25 nt long.

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

6. The method of claim 1, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence set forth in SEQ ID NO:

441.

7. The method of claim 1, wherein the targeter-RNA and/or the activator-RNA comprises one or more of:

a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase.

8. The method of claim 1, wherein the targeter-RNA and/or the activator-RNA comprises one or more of:

(i) a non-natural internucleoside linkage selected from a phosphorothioate, an inverted polarity linkage, and an abasic nucleoside linkage;

(ii) a locked nucleic acid (LNA); and

(iii) a modified sugar moiety selected from 2′

-O-methoxyethyl, 2′

-O-methyl, and 2′

-fluoro.

9. The method of claim 1, wherein the targeter-RNA and/or the activator-RNA comprises one or more of:

a peptide nucleic acid (PNA), a morpholino nucleic acid, a cyclohexenyl nucleic acid (CeNA), and/or a locked nucleic acid (LNA).

10. The method of claim 1, wherein the targeter-RNA and/or the activator-RNA comprises one or more modified sugar moieties selected from:

2′

-O-(2-methoxyethyl), 2′

-dimethylaminooxyethoxy, 2′

-dimethylaminoethoxyethoxy, 2′

-O-methyl, and 2′

-fluoro.

11. The method of claim 1, wherein the targeter-RNA and/or the activator-RNA is conjugated to a moiety selected from:

a polyamine;

a polyamide;

a polyethylene glycol;

a polyether;

a cholesterol moiety;

a cholic acid;

a thioether;

a thiocholesterol;

an aliphatic chain;

a phospholipid;

an adamantane acetic acid;

a palmityl moiety;

an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety;

a biotin;

a phenazine;

a folate;

a phenanthridine;

an anthraquinone;

an acridine;

a fluorescein;

a rhodamine;

a fluor; and

a coumarin.