Scarless genome editing through two-step homology directed repair

A method for scarless editing of genomic DNA of a cell, the method comprising:

• a) performing a first Cas9-mediated homology directed repair (HDR) step comprising:
• i) introducing a first donor polynucleotide into the cell, wherein the first donor polynucleotide comprises a first left homology arm and a first right homology arm for targeting the first donor polynucleotide to a target sequence in the genomic DNA of the cell, wherein the first left and right homology arms flank a sequence comprising an expression cassette encoding at least one selection marker and optionally an intended edit to the target sequence in the genomic DNA;
• and
• ii) introducing a first guide RNA (gRNA) and Cas9 into the cell, wherein the first guide RNA comprises a sequence complementary to the target sequence to be modified in the genomic DNA of the cell, such that the Cas9 forms a first complex with the first guide RNA, said guide RNA directing the first complex to the target sequence, wherein the Cas9 creates a double-stranded break (DSB) at the target sequence in the genomic DNA of the cell, wherein the sequence flanked by the first left and right homology arms in the first donor polynucleotide is integrated into the genomic DNA at the target sequence by Cas9-mediated HDR to produce a genetically modified cell;
• b) isolating the genetically modified cell based on positive selection for the at least one selection marker;
• c) performing a second Cas9-mediated homology directed repair (HDR) step comprising:
• i) introducing a second donor polynucleotide into the genetically modified cell of step (b), wherein the second donor polynucleotide comprises a second left homology arm and a second right homology arm for targeting the second donor polynucleotide to a target sequence in the genomic DNA of the genetically modified cell, wherein the second left and right homology arms flank a sequence which does not comprise the expression cassette encoding the at least one selection marker, and further comprises the intended edit when the sequence flanked by the first left and right homology arms in the first donor polynucleotide in step (a) does not comprise the intended edit;
• ii) introducing a second guide RNA (gRNA) and Cas9 nuclease into the genetically modified cell of step (b), wherein the Cas9 forms a second complex with the second guide RNA, said second guide RNA directing the second complex to the target sequence in the genomic DNA of the genetically modified cell, wherein the Cas9 creates a double-stranded break (DSB) at the target sequence in the genomic DNA, and the sequence flanked by the second left and right homology arms in the second donor polynucleotide is integrated into the genomic DNA at the target sequence by Cas9-mediated HDR, thereby replacing the expression cassette encoding the at least one selection marker from the genomic DNA by Cas9-mediated HDR to produce a genetically modified cell;
• and
• d) isolating the genetically modified cell comprising the intended edit based on negative selection for the at least one selection marker, wherein the expression cassette encoding the at least one selection marker is deleted.