Multiplex production and barcoding of genetically engineered cells

A method for localizing a donor polynucleotide to a genomic target locus in a cell, the method comprising:

• (a) transfecting a cell with a recombinant polynucleotide, the recombinant polynucleotide comprising a genome editing cassette comprising a donor polynucleotide and a DNA binding sequence known to bind a DNA binding domain;
• (b) introducing a nuclease into the cell, wherein the nuclease recognizes and causes a DNA break at the genomic target locus;
• (c) introducing a donor recruitment protein into the cell, the donor recruitment protein comprising the DNA binding domain and a DNA break site localizing domain and allowing the donor recruitment protein to selectively recruit the DNA break, thereby localizing the donor polynucleotide to the genomic target locus.

A library of gene editing vectors, each gene editing vector comprising a genome editing cassette comprising (i) a barcode, (ii) a first nucleic acid sequence encoding a first guide RNA (gRNA) capable of hybridizing at a genomic target locus to be modified, and (iii) a donor polynucleotide, thereby forming a barcode-gRNA-donor polynucleotide combination;

• wherein each recombinant polynucleotide comprises a different genome editing cassette comprising a different barcode-gRNA-donor polynucleotide combination.

A gene editing vector comprising a donor polynucleotide and a first nucleic acid sequence encoding a first guide RNA (guide X) capable of hybridizing with the vector at a target site such that when the guide X is expressed by a cell, the guide X hybridizes to the vector and creates a double-strand DNA break at the target site.