Methods for generating barcoded combinatorial libraries

Patent metadata

Field	Value
Publication number	US-10017760-B2
Application number	US-201715632222-A
Country	US
Kind code	B2
Filing date	Jun 23, 2017
Priority date	Jun 24, 2016
Publication date	Jul 10, 2018
Grant date	Jul 10, 2018

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.

First claim

What is claimed is:

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.

Assignees

• Univ Colorado Regents
• Inscripta Inc

Inventors

• Gill Ryan T
• Garst Andrew
• LIPSCOMB Tanya Elizabeth Warnecke
• Bassalo Marcelo Colika
• ZEITOUN Ramsey Ibrahim

Classifications

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/1079

  Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086) · CPC title

• C12N15/11

  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 title