CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging does not cause heterochromatinization of endogenous loci

We recently developed an easy, efficient and scalable method for tagging and live cell imaging of non-repetitive, endogenous chromosome regions via CRISPR/Cas9 mediated knock-in of a TetO repeat. For this purpose, we created optimized and irregular 48-mer and 96-mer TetO repeats. Since it is known that repetitive regions in the human genome can induce H3K9me3-mediated heterochromatin formation, we tested whether 48-mer and/or 96-mer TetO repeats induce H3K9me3 flanking their insertion sites. Using a newly developed method called as CUT&RUN, we showed that there was no significant difference in the H3K9me3 pattern flanking the insertion sites of TetO repeats when compared to wild-type cells. We performed CUT&RUN using an antibody specific for H3K9me3 to identify H3K9me3 enriched regions in the genome. We controlled H3K9me3 enrichment flanking one of the target sites we named as ‘region 3’ (gRNA target site is chr6:31,813,898-31,813,920 (hg19)), where we integrated TetO repeats by CRISPR/Cas9-mediated knock-in. We compared clones with 96-mer or 48-mer TetO integration at ‘region 3’ (clones 96-8 and 48-15, respectively) versus wild-type cells. We used non-transduced wild-type cells (wt) and TetR-EGFP transduced wild-type (wtGFP) as controls. The experimental samples analyzed were 96-8 and 48-15 clones transduced with TetR-EGFP. Experiments were performed in duplicates (8 samples total).