Nucleic acid-guided nucleases

• US 9,982,279 B1
• Filed: 06/23/2017
• Issued: 05/29/2018
• Est. Priority Date: 06/23/2017
• Status: Active Grant

• Current Assignee: Inscripta, Inc.
• Original Assignee: Inscripta, Inc.
• Inventors: Gill, Ryan T., Garst, Andrew, Lipscomb, Tanya Elizabeth Warnecke
• Primary Examiner(s): Wu, Julie
• Application Number:
  US15/632,001
• Time in Patent Office: 340 Days
• Field of Search: None
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 2319/85 containing an RNA binding domain
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• 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 15/81 for yeasts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/905 in yeast
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2800/22 Vectors comprising a coding region that has been codon optimised for expression in a respective host
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Disclosed herein are engineered non-naturally occurring nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Targetable nuclease systems can be used to edit genetic targets, including recursive genetic engineering and trackable genetic engineering methods.

Claims

1. A nucleic acid-guided nuclease system comprising:

(a) a nucleic acid-guided nuclease comprising the amino acid sequence of SEQ ID NO;

7, wherein the nucleic-acid guided nuclease is encoded by a wild-type or a codon-optimized nucleic acid;

(b) an engineered guide nucleic acid capable of complexing with the nucleic acid-guided nuclease, wherein the engineered guide nucleic acid comprises any one of SEQ ID NO;

88, SEQ ID NO;

93, and SEQ ID NO;

94, and

(c) an editing sequence having a change in sequence relative to the sequence of a target region in a genome of a cell and a mutation in a protospacer adjacent motif (PAM) site;

wherein the system results in a genome edit in the target region in the genome of the cell facilitated by the nuclease, the engineered guide nucleic acid, and the editing sequence.

2. The system of claim 1, wherein the engineered guide nucleic acid and the editing sequence are provided as a single nucleic acid.

3. The system of claim 1, wherein the codon optimized nucleic acid is codon optimized for E. coli.

4. The system of claim 1, wherein the codon optimized nucleic acid is codon optimized for S. cerevisiae.

5. The system of claim 1, wherein the codon optimized nucleic acid is codon optimized for mammalian cells.

6. The system of claim 1, wherein the nucleic acid-guided nuclease has less than 40% protein identity to SEQ ID NO:

12 and SEQ ID NO;

108.

7. The system of claim 1, wherein the codon optimized nucleic acid is codon optimized for plant cells.

8. The system of claim 1, wherein the wild-type nucleic acid comprises the sequence of SEQ ID NO:

27.

9. The system of claim 1, wherein the codon-optimized nucleic acid has at least 95% identity to any one of SEQ ID NO:

47, SEQ ID NO;

133, or SEQ ID NO;

153.

10. A nucleic acid-guided nuclease system comprising:

(a) a nucleic acid comprising SEQ ID NO;

27, SEQ ID NO;

47, SEQ ID NO;

133, or SEQ ID NO;

153, wherein the nucleic acid encodes for a nucleic acid-guided nuclease comprising the amino acid sequence of SEQ ID NO;

7;

(b) an engineered guide nucleic acid capable of complexing with the nucleic acid-guided nuclease, and

(c) an editing sequence having a change in sequence relative to the sequence of a target region in a genome of a cell and a mutation in a protospacer adjacent motif (PAM) site;

wherein the system results in a genome edit in the target region in the genome of the cell facilitated by the nuclease, the engineered guide nucleic acid, and the editing sequence.

11. The system of claim 10, wherein the engineered guide nucleic acid and the editing sequence are provided as a single nucleic acid.

12. The system of claim 10, wherein the nucleic acid encoding for the nucleic acid-guided nuclease is codon optimized for E. coli.

13. The system of claim 10, wherein the nucleic acid encoding for the nucleic acid-guided nuclease is codon optimized for S. cerevisiae.

14. The system of claim 10, wherein the nucleic acid encoding for the nucleic acid-guided nuclease is codon optimized for mammalian cells.

15. The system of claim 10, wherein the nucleic acid encoding for the nucleic acid-guided nuclease is codon optimized for plant cells.

16. The system of claim 10, wherein the nucleic acid-guided nuclease has less than 40% protein identity to SEQ ID NO:

12 and SEQ ID NO;

108.

17. The system of claim 10, wherein the engineered guide nucleic acid comprises any one of SEQ ID NO:

88, SEQ ID NO;

93, SEQ ID NO;

94, and SEQ ID NO;

95.