Methods for generating barcoded combinatorial libraries
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.
1. 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 homologous to a first target nucleic acid;
b) a first site conferring immunity to nuclease-mediated editing; and
c) a first guide nucleic acid sequence homologous to a target region of 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 homologous to a second target nucleic acid, wherein the first 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 homologous to a third target nucleic acid;
b) a second site conferring immunity to nuclease-mediated editing; and
c) a third guide nucleic acid sequence homologous to a target region of the third target nucleic acid, and
iv) 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 homologous to 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.
2. The library of synthesized editing and recorder cassettes of claim 1 , wherein the first guide nucleic acid and the second guide nucleic acid are compatible with a same nucleic acid-guided nuclease.
3. The library of synthesized editing and recorder cassettes of claim 1 , wherein the third guide nucleic acid and the fourth guide nucleic acid are compatible with a same nucleic acid-guided nuclease.
4. The library of synthesized editing and recorder cassettes of claim 1 , wherein the library comprises at least 100, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 total editing and recorder cassettes.
5. The library of synthesized editing and recorder cassettes of claim 4 , wherein the library comprises at least 100, 10 4 , 10 5 , or 10 6 total editing and recorder cassettes.
6. The library of synthesized editing and recorder cassettes of claim 1 , wherein each of the editing cassettes and each of the recorder cassettes is approximately 50-200 nucleotides in length.
7. The library of synthesized editing and recorder cassettes of claim 6 , wherein each of the editing cassettes and each of the recorder cassettes is approximately 75-150 nucleotides in length.
8. The library of synthesized editing and recorder cassettes of claim 7 , wherein each of the editing cassettes and each of the recorder cassettes is approximately 80-120 nucleotides in length.
9. The library of synthesized editing and recorder cassettes of claim 1 , wherein the length of one or both of the barcodes is 4 to 30 nucleotides in length.
10. The library of synthesized editing and recorder cassettes of claim 1 , wherein one or both of the recorder cassettes comprises a landing site.
11. The library of synthesized editing and recorder cassettes of claim 10 , wherein the landing site is an unique sequence.
12. The library of synthesized editing and recorder cassettes of claim 1 , wherein the landing site about 2-50 nucleotides in length.
13. The library of synthesized editing and recorder cassettes of claim 12 , wherein the landing site about 4-20 nucleotides in length.
14. The library of synthesized editing and recorder cassettes of claim 1 , wherein the library of synthesized editing and recorder cassettes promotes edits in bacterial cells.
15. The library of synthesized editing and recorder cassettes of claim 1 , wherein the library of synthesized editing and recorder cassettes promotes edits in eukaryotic cells.
16. The library of synthesized editing and recorder cassettes of claim 1 , wherein the library of synthesized editing and recorder cassettes promotes edits in archeal cells.
17. The library of synthesized editing and recorder cassettes of claim 1 , wherein nucleotide sequences for the synthesized editing and recorder cassettes are designed by software.
18. The library of synthesized editing and recorder cassettes of claim 1 , wherein the synthesized editing and recorder cassettes are pooled.
19. The library of synthesized editing and recorder cassettes of claim 18 , wherein the pooled synthesized editing and recorder cassettes are amplified and cloned in parallel.
20. The library of synthesized editing and recorder cassettes of claim 1 , further comprising a third synthesized editing cassette and a third synthesized recorder cassette, wherein:
i) the third synthesized editing cassette comprises:
a) a modified fourth target nucleic acid sequence homologous to a fourth target nucleic acid;
b) a third site conferring immunity to nuclease-mediated editing; and
c) a fifth guide nucleic acid sequence homologous to a target region of the fourth target nucleic acid; and
ii) the third synthesized recorder cassette comprises:
a) a third barcode for tracking and identifying the modified fourth target nucleic acid sequence; and
b) a six guide nucleic acid sequence homologous to a target region of a second target nucleic acid, wherein the sixth guide nucleic acid sequence is designed to incorporate the third barcode proximal to the second barcode in the barcode array.