CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS

• US 20170175142A1
• Filed: 07/22/2016
• Published: 06/22/2017
• Est. Priority Date: 12/12/2012
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology, Rockefeller University
• Original Assignee: Massachusetts Institute of Technology, Rockefeller University, The Broad Institute, Inc.
• Inventors: MARRAFFINI, Luciano, ZHANG, Feng
• Application Number:
  US15/217,489
• Publication Number:
  US 20170175142A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 38/43 Enzymes; Proenzymes; Derivatives thereof
• CPC Class Codes: A61K 38/46 Hydrolases (3)
• CPC Class Codes: A61K 38/47 acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• 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 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 15/85 for animal cells
• 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 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 2800/90 Vectors containing a transposable element
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/68 : involving nucleic acids

Abstract

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. 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 utilizing the CRISPR-Cas system.

Claims

1-50. -50. (canceled)

51. A method of selecting one or more prokaryotic cell(s), comprising:

(a) introducing to a population of prokaryotic cells an engineered, programmable, non-naturally occurring CRISPR-Cas system comprising a Cas protein and one or more guide RNAs that target genomic loci of DNA molecules encoding one or more gene products in prokaryotic cell(s) of the population, wherein the one or more guide RNAs comprise a guide sequence, a tracr sequence, and a tracr mate sequence, and wherein the Cas protein and the one or more guide RNAs do not naturally occur together;

(b) allowing the Cas protein to cleave the genomic loci of the DNA molecules encoding the one or more gene products,

wherein cleavage of the genomic loci induces cell death, thereby allowing one or more prokaryotic cell(s) having one or more mutations in the genomic loci to be selected.

52. The method of claim 51, wherein the Cas protein is a type II CRISPR system Cas protein.

53. The method of claim 52, wherein the Cas protein is Cas9 enzyme.

54. The method of claim 53, wherein the Cas9 enzyme is a Streptococcus pyogenes Cas9 enzyme.

55. The method of claim 51, wherein the prokaryotic cells are S. pneumoniae.

56. The method of claim 51, wherein the one or more guide RNAs target an antibiotic resistance gene.