Affinity tag for recombination protein recruitment

Abstract

The present disclosure provides compositions and methods to increase the percentage of edited cells in a cell population when employing nucleic-acid guided editing, as well as automated multi-module instruments for performing these methods. Specifically, the disclosure relates to methods, compositions, modules and automated multi-module cell processing instruments that increase the efficiency of nucleic acid-guided editing in a cell population using a nucleic acid nuclease (i.e., an RNA-guided nuclease or “RGN”)/single-strand binding protein (“SSB”) fusion system. The system leverages a single-strand binding protein (SSP) and single-strand DNA annealing protein (“SSAP”) interactions to drive enhanced recruitment.

We claim: 
     
         1 . A system for homologous recombination-based editing of live cells comprising:
 a. a fusion protein comprising two domains:
 i. an N-terminal RNA-guided endonuclease domain; and 
 ii. a C-terminal domain consisting of a full or C-terminal portion of a SSB protein; 
   b. a single-strand DNA annealing protein (SSAP) that is co-expressed with the fusion protein and exhibits high affinity binding for the SSB fusion domain;   c. auxiliary exonuclease proteins that have co-evolved with the SSAP protein; and   d. an editing cassette comprising a gRNA transcription sequence and a repair template transcription sequence present in an editing vector backbone.   
     
     
         2 . The system of  claim 1 , wherein the endonuclease is selected from the group consisting of MAD7, MAD2, MAD4, Cas9, or Cas12. 
     
     
         3 . The system of  claim 2 , wherein the endonuclease is MAD7. 
     
     
         4 . The system of  claim 2 , wherein the endonuclease is MAD4. 
     
     
         5 . The system of  claim 2 , wherein the endonuclease is MAD2. 
     
     
         6 . The system of  claim 2 , wherein the endonuclease is Cas9. 
     
     
         7 . The system of  claim 2 , wherein the endonuclease is Cas12. 
     
     
         8 . The system of  claim 1 , wherein the live cells are mammalian cells, bacteria cells, or yeast cells. 
     
     
         9 . The system of  claim 8 , wherein the live cells are mammalian cells. 
     
     
         10 . The system of  claim 8 , wherein the live cells are bacteria cells. 
     
     
         11 . The system of  claim 1 , wherein the SSB protein is EcSSB and the SSAP is Redβ; the SSB protein is EcSSB and the SSAP is PapRecT; the SSB protein is PaSSB and the SSAP is Redβ; the SSB protein is PaSSB and the SSAP is PapRecT; the SSB protein is MsSSB and the SSAP is MspRecT; the SSB protein is MsSSB and the SSAP is PapRecT; the SSB protein is LrSSB and the SSAP is LrRecT; or the SSB protein is RPA1 and the SSAP is RAD51. 
     
     
         12 . A system for homologous recombination-based editing of live cells comprising:
 a. an RNA-guided endonuclease domain;   b. an over-expressed SSB protein;   c. a single strand DNA annealing protein (SSAP) that is co-expressed with the RGN endonuclease and over-expressed SSB protein and wherein the SSAP exhibits high affinity binding for the SSB;   d. auxiliary exonuclease proteins that have co-evolved with the SSAP protein; and   e. an editing cassette comprising a gRNA transcription sequence and a repair template transcription sequence present in an editing vector backbone.   
     
     
         13 . The system of  claim 12 , wherein the endonuclease is selected from the group consisting of MAD7, MAD2, MAD4, Cas9, or Cas12. 
     
     
         14 . The system of  claim 13 , wherein the endonuclease is MAD7. 
     
     
         15 . The system of  claim 13 , wherein the endonuclease is MAD4. 
     
     
         16 . The system of  claim 13 , wherein the endonuclease is MAD2. 
     
     
         17 . The system of  claim 13 , wherein the endonuclease is Cas9. 
     
     
         18 . The system of  claim 12 , wherein the SSB protein is EcSSB and the SSAP is Redβ; the SSB protein is EcSSB and the SSAP is PapRecT; the SSB protein is PaSSB and the SSAP is Redβ; the SSB protein is PaSSB and the SSAP is PapRecT; the SSB protein is MsSSB and the SSAP is MspRecT; the SSB protein is MsSSB and the SSAP is PapRecT; the SSB protein is LrSSB and the SSAP is LrRecT; or the SSB protein is RPA1 and the SSAP is RAD51. 
     
     
         19 . The system of  claim 12 , wherein the live cells are mammalian cells, bacteria cells, or yeast cells. 
     
     
         20 . The system of  claim 19 , wherein the live cells are mammalian cells. 
     
     
         21 . The system of  claim 19 , wherein the live cells are bacteria cells. 
     
     
         22 . The system of  claim 19 , wherein the live cells are yeast cells. 
     
     
         23 . A system for homologous recombination-based editing of live cells comprising:
 a. a fusion protein comprising two domains:
 i. an N-terminal RNA guided nuclease domain; and 
 ii. a C-terminal domain consisting of a full or C-terminal portion of an SSB protein; 
   b. a single strand DNA annealing protein (SSAP) that is co-expressed with the fusion protein and exhibits high affinity binding for the SSB fusion domain; and   c. an editing cassette comprising a gRNA transcription sequence and a repair template transcription sequence present in an editing vector backbone.   
     
     
         24 . The system of  claim 23 , wherein the endonuclease is selected from the group consisting of MAD7, MAD2, MAD4, Cas9, or Cas12. 
     
     
         25 . The system of  claim 24 , wherein the endonuclease is MAD7. 
     
     
         26 . The system of  claim 24 , wherein the endonuclease is MAD2. 
     
     
         27 . The system of  claim 24 , wherein the endonuclease is Cas9. 
     
     
         28 . The system of  claim 23 , wherein the live cells are mammalian cells, bacteria cells, or yeast cells. 
     
     
         29 . The system of  claim 23 , wherein the SSB protein is EcSSB and the SSAP is Redβ; the SSB protein is EcSSB and the SSAP is PapRecT; the SSB protein is PaSSB and the SSAP is Redβ; the SSB protein is PaSSB and the SSAP is PapRecT; the SSB protein is MsSSB and the SSAP is MspRecT; the SSB protein is MsSSB and the SSAP is PapRecT; the SSB protein is LrSSB and the SSAP is LrRecT; or the SSB protein is RPA1 and the SSAP is RAD51.