Fig. 2: H4 tail lysine saturation mutagenesis and multiplex fitness-profiling.
From: Precision-edited histone tails disrupt polycistronic gene expression controls in trypanosomes
a The alignment shows the N-terminal tail sequence of T. brucei histone H4, compared to the equivalent sequences from yeast and human. Lysine residues are highlighted in the T. brucei sequence. b Schematic of the saturation mutagenesis and amplicon-sequencing strategy. An sgRNA cassette targeting the 5′ end of the H4ECT gene was introduced into an histoneH4 strain. Cas9 was induced, and 24 h later the cells were transfected with single-stranded oligodeoxynucleotide (ssODN) editing templates. DNA was extracted at different timepoints, the edited region of the H4ECT gene was PCR-amplified, and the amplicons were deep-sequenced. Created in BioRender. Novotna, M. (2025) https://BioRender.com/21f7d0g. c The radial plots show the abundance of sequence-reads representing each codon at each edited position during the time course. Data for six edited N-terminal tail lysine’s are shown. Values represent the averages of duplicate samples and are relative to codon scores obtained at the 12 h timepoint. D2, day-2; D4, day-4; D6, day-6. *, stop codons. Source data are provided as a Source Data file. d The logo shows relative overrepresentation or underrepresentation at day-6 for those cognate amino acids that were either tolerated or not tolerated following editing at each of the six lysine positions targeted.
