Figure 5
From: A CRISPR-Cas9 based shuffle system for endogenous histone H3 and H4 combinatorial mutagenesis
Mutagenesis on both histone H3 and H4 through single transformation. (A,B) Graphic representation of CRISPR-Cas9 based gene replacement for mutagenesis on both histone H3 and H4 with different combinations. By transforming different donors, the endogenous two copies of histone H3 and H4 can be repaired for identical mutants (A) or with different mutation sites (B). (C) The mutagenesis efficiencies for generating histone H3 and H4 mutants through single transformation with different gRNAs. 2 gRNAs: 1 gRNA targeting synH3 and 1 gRNA targeting synH4 and 4 gRNAs: 2 gRNAs for each synthetic histone. The data of bar charts represent mean averages of over-all mutagenesis efficiencies of 3 biological replicates with each randomly picked 8 colonies. The error bars indicate the standard deviations of 3 biological replicates. (D) Confirmation of HU sensitivity of histone H3 and H4 mutant. Two biological replicates were grown in YPD overnight at 30 °C. Serial tenfold dilutions were spotted onto YPD or YPD with 100 mM HU and incubated at 30 °C for 3 days.
