Cascade/dCas3 Complementation Assays for In Vivo Detection of Nucleic Acid-Guided Nuclease Edited Cells

Abstract

The present disclosure relates to methods and compositions that allow one to identify in vivo edited cells when employing nucleic-acid guided editing. Additionally provided are automated multi-module instruments for performing editing and selection methods and using the compositions.

We claim: 
     
         1 . A method for in vivo identification of cells properly edited using a Type I CRISPR nucleic acid-guided nuclease split protein editing system, comprising the steps of:
 transforming the cells with a Type I CRISPR nucleic acid-guided nuclease split protein editing system   comprising:
 a library of discriminating vectors comprising nucleic acid sequences encoding editing discriminating guide RNAs (gRNAs) and donor DNAs homologous to a genomic loci in the cells, the gRNAs configured for hybridization to target sequences in the genome that have been rationally edited by a CRISPR enzyme, but do not hybridize to loci on the genome that have not been rationally edited by the CRISPR enzyme, and wherein the gRNAs are configured for interacting with first and second halves of a split protein reporter system; 
 a first vector comprising a nucleic acid sequence encoding the first half of a split protein reporter system comprising a Cascade-T7 polymerase (“Cascade-T7-RNAP) fusion protein, wherein the Cascade-T7-RNAP fusion protein is a Cascade complex fused to C-terminal amino acids of a T7-RNAP or N-terminal amino acids of the T7-RNAP; 
 a second vector nucleic acid sequence encoding the second half of the split protein reporter system comprising a dCas3-T7 RNA polymerase (“dCas-T7-RNAP”) fusion protein, wherein the dCas-T7-RNAP fusion protein coding sequence comprises a dCas3 protein fused to the N-terminal amino acids of the T7-RNAP when the Cascade-T7-RNAP fusion protein comprises C-terminal the amino acids of the T7-RNAP and wherein the dCas-T7-RNAP fusion protein coding sequence comprises a dCas3 protein fused to the C-terminal amino acids of the T7-RNAP when the Cascade-T7-RNAP fusion protein comprises the N-terminal amino acids of the T7-RNAP; and 
 a third vector comprising coding sequence for a reporter gene under the control of a T7 promoter; 
   allowing the Type I CRISPR nucleic acid-guided nuclease split protein editing system to edit the genomic loci in the cells;   providing conditions for the T7 RNAP to bind and activated the T7 promoter thereby transcribing the reporter gene; and   identifying the cells expressing the reporter gene.   
     
     
         2 . The method for in vivo identification of cells of  claim 1 , wherein the first, second and third vectors are combined on the same vector. 
     
     
         3 . The method for in vivo identification of cells of  claim 1 , wherein the first and second vectors are combined on the same vector and the third vector is a separate vector. 
     
     
         4 . The method for in vivo identification of cells of  claim 1 , wherein one or more the target sequences in the genome that have not been rationally edited by the CRISPR enzyme are unedited target sequences. 
     
     
         5 . The method for in vivo identification of cells of  claim 1 , wherein one or more the target sequences in the genome that have not been rationally edited by the CRISPR enzyme are incorrectly edited target sequences. 
     
     
         6 . The method for in vivo identification of cells of  claim 1 , wherein the incorrectly edited target sequences may comprise an indel. 
     
     
         7 . The method for in vivo identification of cells of  claim 1 , wherein the difference between the loci on the genome of the live cells that have been rationally edited by the CRISPR enzyme and the target sequences in the genome that have not been rationally edited by the CRISPR enzyme is at least 5 base pairs of the genomic target sequence. 
     
     
         8 . The method for in vivo identification of cells of  claim 7 , wherein the difference between the loci on the genome of the live cells that have been rationally edited by the CRISPR enzyme and the target sequences in the genome that have not been rationally edited by the CRISPR enzyme is 1 base pair of the genomic target sequence. 
     
     
         9 . The method for in vivo identification of cells of  claim 1 , wherein the Type I CRISPR system is a Type I-C system comprising one or more of Cas5c, Cas7c, or Cas8c. 
     
     
         10 . The method for in vivo identification of cells of  claim 1 , wherein the Type I CRISPR system is a Type I-E system comprising one or more of CasA/Cse1, Cse2, Cas7e, Cas5e, or Cas6e. 
     
     
         11 . The method for in vivo identification of cells of  claim 1 , wherein the first and second halves of the split protein reporter system form an R-loop complex with the RNA guide and target DNA strand in a cell. 
     
     
         12 . The method for in vivo identification of cells of  claim 1 , wherein the Cascade-T7-RNAP fusion protein comprises the C-terminus of the Cas5c protein. 
     
     
         13 . The method for in vivo identification of cells of  claim 1 , wherein the Cascade-T7-RNAP fusion protein comprises the C-terminus of the casA/cse1 protein. 
     
     
         14 . The method for in vivo identification of cells of  claim 1 , wherein the reporter gene codes for a fluorescent protein. 
     
     
         15 . The method for in vivo identification of cells of  claim 14 , wherein the fluorescent protein is a green fluorescent protein or a blue fluorescent protein. 
     
     
         16 . The method for in vivo identification of cells of  claim 1 , wherein the reporter gene codes for a firefly luciferase or a Renilla luciferase. 
     
     
         17 . The method for in vivo identification of cells of  claim 16 , wherein the reporter gene codes for a firefly luciferase. 
     
     
         18 . The method for in vivo identification of cells of  claim 1 , wherein the reporter gene codes for a Renilla luciferase. 
     
     
         19 . The method for in vivo identification of cells of  claim 1 , wherein the reporter gene codes for a broccoli RNA aptamer or a spinach RNA aptamer. 
     
     
         20 . The method for in vivo identification of cells of  claim 19 , wherein the reporter gene codes for a broccoli RNA aptamer. 
     
     
         21 . The Type I CRISPR system of  claim 19 , wherein the reporter gene codes for a spinach RNA aptamer. 
     
     
         22 . A cell transformed with the method for in vivo identification of cells of  claim 1 . 
     
     
         23 . The cell of  claim 22 , wherein the reporter gene is a fluorescent reporter gene.