Figure 2
From: A CRISPR-Cas9 based shuffle system for endogenous histone H3 and H4 combinatorial mutagenesis
Generation of master shuffle strains with synthetic H3 and H4 by CRISPR-Cas9. (A) Generation of a master shuffle strain with synthetic H3 and H4 genes (synH3H4) through 2 rounds of CRISPR-Cas9 mediated gene replacements. The efficiencies of 2 rounds are 3/4 (75%) and 4/4 (100%) respectively, which are tested by four randomly picked colonies respectively. (B,C) The nucleotide sequence alignment of endogenous and synthetic H3 and H4. The synthetic H3 and H4 (synH3 and synH4) share same protein sequences but different DNA codons with native H3 and H4 genes. The nucleotides with grey background indicate the identical nucleotide sequences. Dark blue arrows represent gRNA sequences targeting endogenous H3 (HHT1/2); brown arrows represent gRNA sequences targeting endogenous H4 (HHF1/2); magenta arrows represent gRNA sequences targeting synH3 genes, and light blue arrows represent gRNA sequences targeting synH4 genes. The gRNAs targeting synthetic histones are used for generating Histone H3 and H4 mutants in Figs. 3 and 5.
