Methods for generating barcoded combinatorial libraries

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.

What is claimed is: 
     
         1 . A composition comprising:
 i) a first donor nucleic acid 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 comprising a first spacer region complementary to a portion of the first target nucleic acid; and 
   ii) a second donor nucleic acid comprising:
 a) a barcode corresponding to the modified first target nucleic acid sequence; and 
 b) a second guide nucleic acid sequence comprising a second spacer region complementary to a portion of a second target nucleic acid. 
   
     
     
         2 . The composition of  claim 1 , wherein the modified first target nucleic acid sequence comprises at least one inserted, deleted, or substituted nucleic acid compared to a corresponding un-modified first target nucleic acid. 
     
     
         3 . The composition of  claim 1 , wherein the first guide nucleic acid and second guide nucleic acid are compatible with a nucleic acid-guided nuclease. 
     
     
         4 . The composition of  claim 3 , wherein the nucleic acid-guided nuclease is a Type II or Type V Cas protein. 
     
     
         5 . The composition of  claim 3 , wherein the nucleic acid-guided nuclease is a Cas9 homologue or a Cpf1 homologue. 
     
     
         6 . The composition of  claim 1 , wherein the second donor nucleic acid comprises a second PAM mutation. 
     
     
         7 . The composition of  claim 1 , wherein the second donor nucleic acid sequence comprises a regulatory sequence or a mutation to turn a screenable or selectable marker on or off. 
     
     
         8 . The composition of  claim 1 , wherein the second donor nucleic acid sequence targets a unique landing site. 
     
     
         9 . 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 comprises
 1) 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 comprising a spacer region complementary to a portion of the first target nucleic acid and compatible with the nucleic acid-guided nuclease; and 
 
 2) a recorder cassette comprising
 i) a barcode corresponding to the modified first target nucleic acid sequence; and 
 ii) a second guide nucleic acid sequence comprising a second spacer region complementary to a portion of 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.   
     
     
         10 . The method of  claim 9 , further comprising c) sequencing a portion of the barcode, thereby identifying the modified first target nucleic acid that was inserted within the first target nucleic acid in step a). 
     
     
         11 . The method of  claim 9 , wherein the nucleic acid-guided nuclease is a CRISPR nuclease. 
     
     
         12 . The method of  claim 9 , wherein the PAM mutation is not recognized by the nucleic acid-guided nuclease. 
     
     
         13 . The method of  claim 9 , wherein the nucleic acid-guided nuclease is a Type II or Type V Cas protein. 
     
     
         14 . The method of  claim 9 , wherein the nucleic acid-guided nuclease is a Cas9 homologue or a Cpf1 homologue. 
     
     
         15 . The method of  claim 9 , wherein the recorder cassette further comprises a second PAM mutation that is not recognized by the nucleic acid-guided nuclease. 
     
     
         16 . 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 incorporated during a previous round of engineering, wherein the nucleic acid sequence comprises a unique barcode; 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 unique barcode into the unique landing site.   
     
     
         17 . The method of  claim 16 , wherein the nucleic acid sequence further comprises a regulatory sequence that turns transcription of a screenable or selectable marker on or off. 
     
     
         18 . The method of  claim 16 , wherein the nucleic acid sequence further comprises a PAM mutation that is not compatible with the nucleic acid-guided nuclease. 
     
     
         19 . The method of  claim 16 , wherein the nucleic acid sequence further comprises a second unique landing site for subsequent engineering rounds. 
     
     
         20 . The method of  claim 16 , 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 comprising a first spacer region complementary to a portion of the first target nucleic acid,   
       wherein the unique barcode corresponds to the modified first target nucleic acid such that the modified target nucleic acid can be identified by the unique barcode.