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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
First Claim
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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 comprises1) 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 comprisingi) 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.

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