Fig. 1: Complementation of >40 T. brucei histone H4 genes with a recoded H4 gene.

a The schematic shows hist^oneH4 strain construction. The 2T1^T7-Cas9 strain was transfected with a cassette containing a recoded, and ectopic histone H4 gene (H4^ECT, synonymous changes highlighted) and a single guide RNA (sgRNA) targeting H4^NAT genes, both under the control of a T7 RNA polymerase promoter (T7). Cas9 was induced for 24 h in the resulting cell line, which was then transfected with an NPT (neomycin phosphotransferase) cassette to delete the native histone H4 (H4^NAT) tandem arrays. Created in BioRender. Novotna, M. (2025) https://BioRender.com/f0do2k7. b The Southern blots were probed for the 5′ end of the native H4 genes (left), which detected the expected 736 bp band (approx. 43 copies), and 1505 bp band (1 copy from each of 2 alleles) only in the parental cell line. The blot was stripped and re-probed for the ectopic H4 gene (right) revealing the expected 4664 bp and 13803 bp bands in both independent hist^oneH4 strains, but not in the parent. Some residual signal, or cross-reactivity, can be seen from the 736 bp band; the ectopic H4 probe shares 84% sequence identity with native H4 genes. M, digoxigenin (DIG)-labelled DNA ladder. c The circular plot shows whole-genome sequencing data for the hist^oneH4 strains compared to the parental strain, and the parental strain plus the H4^ECT gene. The zoom at top shows precise deletion of the native H4 arrays; clone 1, blue; clone 2, magenta. d RNA-seq analysis. A strain expressing the H4^ECT gene was compared to the parent strain in the upper panels. Both hist^oneH4 strains were compared to the strain expressing the H4^ECT gene in the lower panels. The H4^NAT and H4^ECT transcripts (orange) and the super-abundant VSG transcript are highlighted in the left-hand panels, while five genes immediately downstream of the H4 array are highlighted (blue) in the right-hand panels. n = 8934.