Methods for generating barcoded combinatorial libraries
AssigneesThe Regents of the University of Colorado, a body corporate, Inscripta, Inc.
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.
Claims20
A library of synthesized editing and recorder cassettes, the library comprising:
- a first synthesized editing cassette and a first synthesized recorder cassette, wherein:
- i) the first synthesized editing cassette comprises:
- a) a modified first target nucleic acid sequence targeting a first target nucleic acid;
- b) a first site conferring immunity to nuclease-mediated editing;
- and
- c) a first guide nucleic acid sequence targeting the first target nucleic acid, and
- ii) the first synthesized recorder cassette comprises:
- a) a first 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 a sequence homologous to the second target nucleic acid;
- and
- at least a second synthesized editing cassette and a second synthesized recorder cassette, wherein
- iii) the second synthesized editing cassette comprises:
- a) a modified third target nucleic acid sequence targeting a third target nucleic acid;
- b) a second site conferring immunity to nuclease-mediated editing;
- and
- c) a third guide nucleic acid sequence targeting the third target nucleic acid, and
- ii) the second synthesized recorder cassette comprises
- a) a second barcode for tracking and identifying the modified third target nucleic acid sequence;
- and
- b) a fourth guide nucleic acid sequence targeting a fourth target nucleic acid, wherein the fourth guide nucleic acid sequence is designed to incorporate the second barcode proximal to the first barcode forming a barcode array.
Dependencies are inferred from the claim text.