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

A Type I CRISPR system comprising a library of discriminating vectors and vectors comprising a split protein and a reporter gene for identifying cells in a population of cells, wherein the cells have a genome that has been rationally edited, the Type I CRISPR system comprising:

• the library of discriminating vectors comprising nucleic acid sequences encoding editing discriminating guide RNAs (gRNAs), 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;
• and
• 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.