Figure 3

Integration efficiency of tHMG1 at locus X-2 using different lengths of homology arms. a Overview of the donor DNA fragment bearing tHMG1 with differently sized homology arms. b Integration efficiency of the CrEdit system with genomic inducible Cas9 and integrative gRNA. S. cerevisiae strain ST1011 harboring P _CUP1 -cas9 was induced with Cu²⁺ 2 h prior to transformation start, and then co-transformed with (left, –gRNA) linearized empty vector pCfB257 and linearized donor DNA encoding tHMG1 (for details of donor DNA see Additional file 1), or (right, +gRNA) the linearized integrative gRNA vector pCfB2831 targeting X-2 and linearized donor DNA encoding tHMG1. Left panel Efficiency of targeted integration at site X-2 when selecting for donor DNA after transformation. Middle panel Efficiency of marker gene integration when not selecting for donor DNA after transformation. Right panel Frequency of correct integration at site X-2 determined by genotyping of URA⁺ colonies. c Integration efficiency of the CrEdit system with plasmid-based Cas9 and gRNA. S. cerevisiae strain TC-3 harboring P _TEF1 -cas9 on the centromeric plasmid pCfB1767 was co-transformed with (left, −gRNA) empty vector pCfB2999 and linearized donor DNA encoding tHMG1, or (right, +gRNA) the episomal gRNA vector pCfB3020 targeting X-2 and linearized donor DNA encoding tHMG1. Left panel Efficiency of targeted integration at site X-2 when selecting for donor DNA after transformation. Middle panel Efficiency of marker gene integration when not selecting for donor DNA after transformation. Right panel Frequency of correct integration at site X-2 determined by genotyping of URA⁺ colonies. Only +gRNA colonies were analyzed since no URA ⁺ clones were obtained in the absence of gRNA. The experiment was repeated twice and error bars represent 95% confidence intervals. NA not analyzed.