Figure 1. Germline knock-in of H3.3G34R/V/W results in distinct developmental defects.

A. Left: amino acid depiction of H3.3 glycine 34 (G, hydrophobic) substitution to arginine (R, basic), valine (V, hydrophobic) or tryptophan (W, aromatic). Right: schematic representation of the derivation of H3.3G34R/V/W direct knock-in mice. Single-guide RNA (sgRNA) targeting exon 2 of H3f3a is represented by the red box, and the ssODN homology-directed repair (HDR) donor sequence is shown inside the gray box. The HDR template contained the G-to-R/V/W nucleotide substitutions at the G34 codon (red box) and the three silent PAM-blocking mutations (black boxes). Cas9 protein, sgRNA, and ssODN were microinjected into 2- to 4-cell embryos to obtain mosaic founder mice (F0) and backcrossed to two genetic strains to obtain true heterozygous generation 1 (G1) founders. B. Immunohistochemistry staining for H3.3G34R/V/W in DKI mutant mice. C. G34R/V/W mice show distinct and overlapping developmental phenotypes. Right: bar chart depicting penetrance of the observed phenotypes for each G34 mutant, > 10 mice were sampled for each genotype and phenotype combination. D. Body weight of G34 mutant and indel mice represented as ratios normalized to WT mice. n: cumulative number of mice. Representative pictures of WT and G34W mice at P7 and 5 months of age. E. Kaplan-Meier curves for cumulative survival rates of male G34 mutants and controls, n: cumulative number of mice.