Cure all for nucleic acid-guided cell editing in E. Coli

A method for curing cells during recursive nucleic acid-guided nuclease editing or after a last round of nucleic acid-guided nuclease editing comprising:

• designing and synthesizing a library of editing cassettes, wherein the library of editing cassettes comprises at least one editing cassette;
• assembling the library of editing cassettes into a vector backbone thereby forming a library of editing vectors, wherein the vector backbone comprises an inducible promoter to drive transcription of the at least one editing cassette;
• a first selectable marker and a curing target sequence;
• making cells of choice electrocompetent, wherein the cells of choice comprise an engine vector and the engine vector comprises a nuclease under the control of the second inducible promoter and a second selectable marker;
• transforming the cells of choice with the library of editing vectors to produce first transformed cells;
• selecting for first transformed cells via the first or second selectable markers;
• inducing editing in the selected cells by inducing the first and second inducible promoters thereby inducing transcription of the editing cassette and the nuclease thereby producing edited cells;
• growing the edited cells;
• transforming the edited cells with a curing vector to produce transformed edited cells, wherein the curing vector comprises a promoter driving transcription of an anti-curing target gRNA;
• a coding sequence for a nuclease compatible with the anti-curing target gRNA;
• and a coding sequence for a third antibiotic resistance gene, wherein the third antibiotic resistance gene is different from the second antibiotic resistance gene;
• and
• curing the editing and engine vectors by growing the transformed edited cells in medium comprising the third antibiotic and providing conditions to transcribe the anti-curing target gRNA thereby creating cured cells.