| abstract |
A eukaryotic cell for use in Sequential Endonuclease Mediated Homology Directed Recombination (sEHDR) comprising a method of Cas Endonuclease-mediated Recombination, wherein the cell: a) a first strand of DNA comprising first, second and third PAM motifs, the second and third PAM motifs flanking a nucleic acid sequence to be replaced; b) an insertion nucleotide sequence comprising first and second homology arms, wherein the first homology arm is substantially homologous to the sequence extending 5 'from the second PAM motif and the second homology arm is substantially homologous to the sequence extending 3' from the third PAM motif; c) a first guide RNA that hybridizes to the sequence 5 'to the second PAM motif; d) a second guide RNA which hybridizes with the sequence 5 'to the third PAM motif; and e) the Cas endonuclease, unfit, and wherein the first and third PAM motifs can be used for another round of Cas endonuclease-mediated recombination. |