ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS FOR SEQUENCE MANIPULATION

• US 20150232882A1
• Filed: 05/04/2015
• Published: 08/20/2015
• Est. Priority Date: 12/12/2012
• Status: Active 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
• Application Number:
  US14/703,511
• Publication Number:
  US 20150232882A1
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system comprising one or more vectors comprising:

a) a first regulatory element operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system polynucleotide sequences comprising a guide sequence, a tracr RNA, and a tracr mate sequence, wherein the guide sequence hybridizes with one or more target sequences in polynucleotide loci in a eukaryotic cell,

b) a second regulatory element operably linked to a nucleotide sequence encoding a Type II Cas9 protein,

wherein components (a) and (b) are located on same or different vectors of the system,

wherein the CRISPR-Cas system comprises two or more nuclear localization signals (NLSs) expressed with the nucleotide sequence encoding the Cas9 protein,

whereby the one or more guide sequences target the one or more polynucleotide loci in a eukaryotic cell and the Cas9 protein cleaves the one or more polynucleotide loci, whereby the sequence of the one or more polynucleotide loci is modified.

2. An engineered, non-naturally occurring Type II CRISPR-Cas vector system according to claim 1,

wherein the Cas9 protein is mutated with respect to a corresponding wild type Cas9 protein such that the mutated protein is a nickase that lacks the ability to cleave one strand of a target polynucleotide,

whereby the one or more guide sequences target the one or more polynucleotide loci in a eukaryotic cell and the Cas9 protein cleaves only one strand of the polynucleotide loci, whereby the sequence of the one or more polynucleotide loci is modified.

3. The system of claim 1 or 2, wherein the vectors are viral vectors.

4. The system of claim 3, wherein the viral vectors are retroviral, lentiviral, adenoviral, adeno-associated or herpes simplex viral vectors.

5. The system of any of claims 2-4 wherein the Cas9 protein comprises one or more mutations in the RuvC I, RuvC II or RuvC III catalytic domains.

6. The system of any of claims 2-4 wherein the Cas9 protein comprises a mutation selected from the group consisting of D10A, H840A, N854A and N863A with reference to the position numbering of a Streptococcus pyogenes Cas9 (SpCas9) protein.

7. The system of any preceding claim, wherein at least one NLS is at or near amino-terminus of the Cas9 protein and/or at least one NLS is at or near carboxy terminus of the Cas9 protein.

8. The system of claim 7, wherein at least one NLS is at or near amino-terminus of the Cas9 protein and at least one NLS is at or near carboxy terminus of the Cas9 protein.

9. The system of any preceding claim, wherein the one or more CRISPR-Cas system polynucleotide sequences comprise a guide sequence fused to a trans-activating cr (tracr) sequence.

10. The system of any preceding claim, wherein the CRISPR-Cas system polynucleotide sequence is a chimeric RNA comprising the guide sequence, the tracr sequence, and a tracr mate sequence.

11. The system of any preceding claim, wherein the eukaryotic cell is a mammalian cell or a human cell.

12. The system of any preceding claim, wherein the Cas9 protein is codon optimized for expression in a eukaryotic cell.

13. Use of the system of any of claims 1 to 12 for genome engineering, provided the use does not comprise a process for modifying the germ line genetic identity of human beings, and provided that said use is not a method for treatment of the human or animal body by surgery or therapy.

14. The use of claim 13 wherein the genome engineering comprises modifying a target polynucleotide in a eukaryotic cell, modifying expression of a polynucleotide in a eukaryotic cell, generating a model eukaryotic cell comprising a mutated disease gene, or knocking out a gene.

15. The use of claim 13 wherein the use further comprises repairing said cleaved target polynucleotide by inserting an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide.

16. The use of claim 13 wherein the use further comprises editing said cleaved target polynucleotide by inserting an exogenous template polynucleotide, wherein said edit results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide.

17. The use of claim 15 or 16 wherein the inserting is by homologous recombination.

18. Use of the system of any of claims 1 to 12 in the production of a non-human transgenic animal or transgenic plant.