Methods for generating barcoded combinatorial libraries

Patent metadata

Field	Value
Publication number	US-10287575-B2
Application number	US-201815948798-A
Country	US
Kind code	B2
Filing date	Apr 9, 2018
Priority date	Jun 24, 2016
Publication date	May 14, 2019
Grant date	May 14, 2019

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:
contacting a population of cells with a polynucleotide, such that at least one cell uptakes a first single molecule polynucleotide comprising a first editing cassette and a first recorder cassette, wherein:
a),
1) the first editing cassette comprises:
i) a modified first target nucleic acid sequence targeting a first target nucleic acid;
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) the first recorder cassette comprises:
i) a first 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;
c) contacting said at least one cell with at least one additional single molecule polynucleotide comprising a second editing cassette and a second recorder cassette, wherein
3) the second editing cassette comprises:
i) a modified third target nucleic acid sequence targeting a third target nucleic acid:
ii) a second protospacer adjacent motif (PAM) mutation; and
iii) a third guide nucleic acid sequence targeting the third target nucleic acid, and

4) the second recorder cassette comprises:
i) a second barcode for tracking and identifying the modified third target nucleic acid sequence; and
ii) 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; and

d) allowing the third guide nucleic acid sequence, the fourth guide nucleic acid sequence, and the nucleic acid-guided nuclease to create a genome edit within the third target nucleic acid and the fourth target nucleic acid,
wherein the modified first target nucleic acid sequence is different to the modified third nucleic acid sequence, and wherein the second barcode is inserted adjacent to the first barcode, thereby forming a barcode array.

2. The method of claim 1 , further comprising e) sequencing the barcodes in a single reaction, thereby identifying the modified first and third target nucleic acids that were inserted within the first target nucleic acid in a) and c).

3. The method of claim 1 , wherein the first PAM mutation is not recognized by the nucleic acid-guided nuclease.

4. The method of claim 1 , wherein the second recorder cassette further comprises a second PAM mutation that is not recognized by the nucleic acid-guided nuclease.

5. The method of claim 1 , wherein the second recorder cassette targets a unique landing site.

6. The method of claim 1 , wherein the second target nucleic acid comprises a unique landing site.

7. The method of claim 1 ,
wherein the nucleic acid-guided nuclease comprises amino acid sequence SEQ ID NO: 7.

8. The method of claim 1 , wherein the nucleic acid-guided nuclease comprises amino acid sequence SEQ ID NO: 2.

9. 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, wherein the nucleic acid sequence comprises a barcode, wherein said barcode is a heterologous sequence engineered into said cells during a first round of said selectable recursive genetic engineering; 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 barcode into the unique landing site;
wherein the nucleic acid-guided nuclease comprises amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 7, and

repeating a) and b) during a second round of said selectable recursive genetic engineering.

10. The method of claim 9 , wherein the nucleic acid sequence further comprises a regulatory sequence that turns transcription of a screenable or selectable marker on or off.

11. The method of claim 9 , wherein the nucleic acid sequence further comprises a PAM mutation that is not compatible with the nucleic acid-guided nuclease.

12. The method of claim 9 , wherein the nucleic acid sequence further comprises a second unique landing site.

13. The method of claim 9 , 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 barcode.

14. The method of claim 13 , wherein the modified first target nucleic acid sequence comprises at least one inserted, deleted, or substituted nucleotide.

15. The method of claim 13 , wherein the modified target nucleic acid sequence is within a coding region of the first target nucleic acid.

16. The method of claim 13 , wherein the modified target nucleic acid sequence is within a non-coding region of the first target nucleic acid.

Assignees

• Univ Colorado Regents
• Inscripta Inc

Inventors

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

Classifications

• 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

• C12N15/102 Primary

  Mutagenizing nucleic acids · 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

• C12N15/1065 Primary

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