METHODS, SYSTEMS, AND APPARATUS FOR IDENTIFYING TARGET SEQUENCES FOR CAS ENZYMES OR CRISPR-CAS SYSTEMS FOR TARGET SEQUENCES AND CONVEYING RESULTS THEREOF

• US 20140186843A1
• Filed: 12/12/2013
• Published: 07/03/2014
• Est. Priority Date: 12/12/2012
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: ZHANG, Feng, HABIB, Naomi
• Application Number:
  US14/104,900
• Publication Number:
  US 20140186843A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• 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 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

Disclosed are locational or positional methods concerning CRISPR-Cas systems, and apparatus therefor.

Claims

1. A method of identifying one or more unique target sequences susceptible to being recognized by a CRISPR-Cas system in a genome of a eukaryotic organism, wherein the method comprises:

locating a CRISPR motif;

analyzing a sequence upstream of the CRISPR motif to determine if the sequence occurs elsewhere in the genome;

selecting the sequence if it does not occur elsewhere in the genome, thereby identifying a unique target site.

2. The method of claim 1, wherein the sequence upstream of the CRISPR motif is at least 20 bp in length.

3. The method of claim 1, wherein the sequence upstream of the CRISPR motif is at least 12 bp in length.

4. The method of claim 1, wherein the sequence upstream of the CRISPR motif is at least 10 bp in length.

5. The method of claim 1, wherein the CRISPR motif is recognized by a Cas9 enzyme.

6. The method of claim 1, wherein the CRISPR motif is recognized by a SpCas9 enzyme.

7. The method of claim 1, wherein the CRISPR motif is NGG.

8. The method of claim 1, wherein the eukaryotic organism is selected from the group consisting of Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (roundworm), Sus scrofa (pig) and Bos taurus (cow).

9. A computer-readable medium comprising codes that, upon execution by one or more processors, implements a method of identifying one or more unique target sequences in a genome of a eukaryotic organism, whereby the target sequence is susceptible to being recognized by a CRISPR-Cas system, wherein the method comprises:

locating a CRISPR motif;

analyzing a sequence upstream of the CRISPR motif to determine if the sequence occurs elsewhere in the genome;

selecting the sequence if it does not occur elsewhere in the genome, thereby identifying a unique target site.

10. The computer-readable medium of claim 9, wherein the sequence upstream of the CRISPR motif is at least 20 bp in length.

11. The computer-readable medium of claim 9, wherein the sequence upstream of the CRISPR motif is at least 12 bp in length.

12. The computer-readable medium of claim 9, wherein the sequence upstream of the CRISPR motif is at least 10 bp in length.

13. The computer-readable medium of claim 9, wherein the CRISPR motif is recognized by a Cas9 enzyme.

14. The computer-readable medium of claim 9, wherein the CRISPR motif is recognized by a SpCas9 enzyme.

15. The computer-readable medium of claim 9, wherein the CRISPR motif is NGG.

16. The computer-readable medium of claim 9, wherein the eukaryotic organism is selected from the group consisting of Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (roundworm), Sus scrofa (pig) and Bos taurus (cow).

17. A computer system for identifying one or more unique target sequences in a genome of a eukaryotic organism, the system comprising:

a. a memory unit configured to receive and/or store sequence information of the genome; and

b. one or more processors alone or in combination programmed to (i) locate a CRISPR motif, (ii) analyze a sequence upstream of the CRISPR motif to determine if the sequence occurs elsewhere in the genome, (iii) select the sequence if it does not occur elsewhere in the genome, thereby identifying a unique target site and (iv) display the one or more unique target sequences.

18. The system claim 17, wherein the sequence upstream of the CRISPR motif is at least 20 bp in length.

19. The system of claim 17, wherein the sequence upstream of the CRISPR motif is at least 12 bp in length.

20. The system of claim 17, wherein the sequence upstream of the CRISPR motif is at least 10 bp in length.

21. The system of claim 17, wherein the CRISPR motif is recognized by a Cas9 enzyme.

22. The system of claim 17, wherein the CRISPR motif is recognized by a SpCas9 enzyme.

23. The system of claim 17, wherein the CRISPR motif is NGG.

24. The system of claim 17, wherein the eukaryotic organism is selected from the group consisting of Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (roundworm), Sus scrofa (pig) and Bos taurus (cow).