Methods for generating barcoded combinatorial libraries
AssigneesInscripta, Inc., The Regents of the University of Colorado, a body corporate
InventorsRyan T. Gill, Andrew Garst, Tanya Elizabeth Warnecke Lipscomb, Marcelo Colika Bassalo, Ramsey Ibrahim Zeitoun
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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.
Claims12
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.
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.
Dependencies are inferred from the claim text.