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US 10,294,473 B2Granted
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 single molecule polynucleotides, the library comprising:
- a first single molecule polynucleotide comprising a first editing cassette and a first recorder cassette, wherein:
- i) the first editing cassette comprises:
- a) a modified first target nucleic acid sequence targeting a first target nucleic acid;
- b) a first protospacer adjacent motif (PAM) mutation;
- and
- c) a first guide nucleic acid sequence targeting the first target nucleic acid, and
- ii) the first 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 one additional single molecule polynucleotide comprising a second editing cassette and a second recorder cassette, wherein
- iii) the second editing cassette comprises:
- a) a modified third target nucleic acid sequence targeting a third target nucleic acid;
- b) a second protospacer adjacent motif (PAM) mutation;
- and
- c) a third guide nucleic acid sequence targeting the third target nucleic acid, and
- ii) the second 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.
19 more elements
Dependencies are inferred from the claim text.