Extended Data Fig. 1: Validation of CRISPR gene editing of H3.3 in ESCs.

a, General scheme used to introduce point-mutations into H3.3B. Guide sequences were designed to target Cas9 to the first coding exon of H3.3B close to the mutation site. The single-stranded DNA repair template (ssODN) contains nucleotide changes to introduce lysine-to-alanine mutation and 3 additional synonymous mutations inside the sequence complementary to the guide or inside the PAM to prevent re-cleavage after repair. Optionally, synonymous mutations can give rise to a new restriction site used to validate clones. b, Example of a restriction digest using a newly introduced restriction site after CRISPR editing. Genomic DNA of wildtype cells and a mutant cell line was used for PCR amplification followed by restriction digest with BanI. Experiments were repeated at least three times independently with similar results. c, Sanger-sequencing results of the H3.3B locus for H3.3K4A and H3.3K36A mutant cells. Blat tool is used to compare the results with the wildtype genome (mm10) and the search is visualized using UCSC. Zoom into the region of the first coding exon reveals successful introduction of lysine-to-alanine mutation at either K4 or K36, respectively, and the introduction of 3 additional synonymous mutations inside guide recognition site or PAM. d, Analysis of chromatograms from Sanger sequencing confirms the homozygous exchange of targeted nucleotides in H3.3K4A/K36A mutant cell lines. e, Normalized RNA-seq counts of H3.3A (H3f3a) expression in controls, H3.3K4A/K36A ESCs in comparison to wild type ESCs, confirming knockout of H3.3A.