METHODS FOR GENERATING BARCODED COMBINATORIAL LIBRARIES

• US 20180230460A1
• Filed: 04/09/2018
• Published: 08/16/2018
• Est. Priority Date: 06/24/2016
• Status: Active Grant

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: Inscripta, Inc., The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: GILL, Ryan T., GARST, Andrew, LIPSCOMB, Tanya Elizabeth Warnecke, BASSALO, Marcelo Colika, ZEITOUN, Ramsey Ibrahim
• Granted Patent: US 10,294,473 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086
• 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 15/11 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 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Provided herein are methods and composition for trackable genetic variant libraries. Further provided herein are methods and compositions for recursive engineering. Further provided herein are methods and compositions for multiplex engineering. Further provided herein are methods and compositions for enriching for editing and trackable engineered sequences and cells using nucleic acid-guided nucleases.

Claims

1. A composition comprising:

i) a first nucleic acid sequence comprising;

a) a modified first target nucleic acid sequence;

b) a first protospacer adjacent motif (PAM) mutation; and

c) a first guide nucleic acid sequence targeting the first target nucleic acid, and

ii) a second nucleic acid sequence comprising;

a) a barcode for tracking and identifying the modified first target nucleic acid sequence; and

b) a second guide nucleic acid sequence targeting a second target nucleic acid,

wherein the barcode is flanked by sequence homologous to the second target nucleic acid.

2. The composition of claim 1, wherein the modified first target nucleic acid sequence comprises at least one inserted, deleted, or substituted nucleotide.

3. The composition of claim 1, wherein the modified target nucleic acid sequence is within a coding region of the first target nucleic acid.

4. The composition of claim 1, wherein the modified target nucleic acid sequence is within a non-coding region of the first target nucleic acid.

5. The composition of claim 1, wherein the first guide nucleic acid and second guide nucleic acid are compatible with a nucleic acid-guided nuclease.

6. The composition of claim 5, wherein the nucleic acid-guided nuclease is a Type II or Type V Cas protein.

7. The composition of claim 5, wherein the nucleic acid-guided nuclease is a Cas9 homologue or a Cpf1 homologue.

8. The composition of claim 5, wherein the nucleic acid-guided nuclease is Mad2.

9. The composition of claim 5, wherein the nucleic acid-guided nuclease comprises SEQ ID NO:

2.

10. The composition of claim 5, wherein the nucleic acid-guided nuclease is Mad7.

11. The composition of claim 5, wherein the nucleic acid-guided nuclease comprises SEQ ID NO:

7.

12. The composition of claim 1, wherein the second donor nucleic acid comprises a second PAM mutation.

13. The composition of claim 1, wherein the second donor nucleic acid comprises a unique landing site.

14. The composition of claim 1, wherein the barcode comprises a unique landing site.

15. The composition of claim 1, wherein the second donor nucleic acid sequence comprises a regulatory sequence or a mutation to turn a screenable or selectable marker on or off.

16. The composition of claim 1, wherein the barcode comprises a regulatory sequence or a mutation to turn a screenable or selectable marker on or off.

17. The composition of claim 1, wherein the second guide nucleic acid sequence targets a unique landing site.

18. The composition of claim 1, wherein the second target nucleic acid comprises a unique landing site.

19. The composition of claim 1, wherein the first or second guide nucleic acid comprises a guide RNA (gRNA).

20. The composition of claim 1, wherein the first or second guide nucleic acid comprises a crRNA.