Methods for generating barcoded combinatorial libraries

• US 10,017,760 B2
• Filed: 06/23/2017
• Issued: 07/10/2018
• 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
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US15/632,222
• Publication Number:
  US 20170369870A1
• Time in Patent Office: 382 Days
• Field of Search: None
• 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 method of genome engineering, the method comprising:

a) contacting a population of cells with a polynucleotide, wherein each cell comprises a first target nucleic acid, a second target nucleic acid, and a nucleic acid-guided nuclease,

wherein the polynucleotide comprises

1) an editing cassette comprising;

i) a modified first target nucleic acid sequence;

ii) a first protospacer adjacent motif (PAM) mutation;

iii) a first guide nucleic acid sequence targeting the first target nucleic acid and compatible with the nucleic acid-guided nuclease; and

2) a recorder cassette comprising

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

ii) a second guide nucleic acid sequence targeting the second target nucleic acid and compatible with the nucleic acid-guided nuclease;

b) allowing the first guide nucleic acid sequence, the second guide nucleic acid sequence, and the nucleic acid-guided nuclease to create a genome edit within the first target nucleic acid and the second target nucleic acid; and

c) using the PAM mutation to enrich for cells comprising the genome edit within the first target nucleic acid and second target nucleic acid.

2. The method of claim 1, further comprising d) sequencing the barcode, thereby identifying the modified first target nucleic acid that was inserted within the first target nucleic acid in step a).

3. The method of claim 1, wherein the nucleic acid-guided nuclease is a CRISPR nuclease.

4. The method of claim 1, wherein the PAM mutation is not recognized by the nucleic acid-guided nuclease.

5. The method of claim 1, wherein the nucleic acid-guided nuclease is a Type II or Type V Cas protein.

6. The method of claim 1, wherein the nucleic acid-guided nuclease is a Cas9 homologue or a Cpf1 homologue.

7. The method of claim 1, wherein the recorder cassette further comprises a second PAM mutation that is not recognized by the nucleic acid-guided nuclease.

8. A method of selectable recursive genetic engineering comprising

a) contacting cells comprising a nucleic acid-guided nuclease with a polynucleotide comprising a recorder cassette, said recorder cassette comprising

i) a nucleic acid sequence that recombines into a unique landing site incorporated during a previous round of engineering, wherein the nucleic acid sequence comprises a unique barcode;

and

ii) a guide RNA compatible with the nucleic acid-guided nuclease that targets the unique landing site; and

b) allowing the nucleic acid-guided nuclease to edit the unique landing site, thereby incorporating the unique barcode into the unique landing site.

9. The method of claim 8, wherein the nucleic acid sequence further comprises a regulatory sequence that turns transcription of a screenable or selectable marker on or off.

10. The method of claim 8, wherein the nucleic acid sequence further comprises a PAM mutation that is not compatible with the nucleic acid-guided nuclease.

11. The method of claim 8, wherein the nucleic acid sequence further comprises a second unique landing site for subsequent engineering rounds.

12. The method of claim 8, wherein the polynucleotide further comprises an editing cassette 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,

wherein the modified first target nucleic acid can be tracked and identified by the unique barcode.