CRISPR-Cas component systems, methods and compositions for sequence manipulation

• US 9,822,372 B2
• Filed: 01/07/2016
• Issued: 11/21/2017
• Est. Priority Date: 12/12/2012
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology, Rockefeller University
• Original Assignee: Massachusetts Institute of Technology, Rockefeller University, The Broad Institute, Inc.
• Inventors: Zhang, Feng, Cox, David Benjamin Turitz, Marraffini, Luciano, Bikard, David Olivier, Jiang, Wenyan, Sanjana, Neville Espi
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US14/990,444
• Publication Number:
  US 20160115488A1
• Time in Patent Office: 684 Days
• Field of Search: None
• 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 selecting one or more prokaryotic cell(s) by introducing one or more mutations in one or more prokaryotic cell(s), the method comprising:

introducing one or more vectors into the prokaryotic cell(s);

wherein the one or more vectors drive expression of;

a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template for recombination into a targeted chromosomal locus comprising a target polynucleotide;

and all of the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and a tracr sequence, and the editing template are produced in the prokaryotic cell(s), whereby a CRISPR complex forms;

the CRISPR complex comprising the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence;

wherein the CRISPR complex binds to the target polynucleotide;

wherein the editing template is introduced into the target polynucleotide through recombination, wherein the editing template comprises one or more mutations of the target polynucleotide that alter either a protospacer-adjacent motif (PAM) sequence or a protospacer sequence, and that abolishes CRISPR enzyme cleavage of the target polynucleotide;

wherein in those cells that have not had the editing template introduced there is cleavage of the target polynucleotide by the CRISPR complex and cell death; and

selecting one or more prokaryotic cell(s) in which the one or more mutations have been introduced.

2. The method of claim 1, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

3. The method of claim 1, wherein the CRISPR enzyme is a Cas9.

4. The method of claim 3, wherein the Cas9 is S. pyogenes Cas9.

5. The method of claim 1 wherein the CRISPR enzyme is not endogenous to the prokaryotic cell(s).

6. The method of claim 1 wherein the prokaryotic cell(s) are S. pneumoniae or E. coli.

7. The method of claim 1 wherein the one or more vectors are plasmids.

8. The method of claim 1 wherein the one or more mutations alter the PAM sequence of the target polynucleotide.