Decorating chromatin for precise genome editing using CRISPR Cas

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Project Summary / Abstract CRISPR associated (Cas) systems have revolutionized biology by allowing the introduction of targeted double stranded DNA breaks (DSB). However, in human cells, non-homologous end joining (NHEJ) is the preferred DSB repair process which often leads to small insertions and deletions (indels) at the site of CRISPR-Cas- induced DSBs. As a result, CRISPR systems are efficient tools for creating genetic knockouts but making targeted insertions remains a challenge. Efficient targeted insertions would allow for quick generation of cancer models and would also pave the way for next generation adoptive T cell therapies, where chimeric antigen receptor (CAR) transgenes could be inserted at specific genomic sites that improve their ability to destroy cancer cells. Homology driven repair (HDR) mediated by donor templates is currently the best way to introduce precise edits and DNA insertions following CRISPR-Cas cutting, but the efficiency is generally low relative to NHEJ, and strategies to improve it have yielded only small advancements. Here, I propose an approach to improve HDR by making chromatin more accessible at the CRISPR-Cas induced cut site and decorating adjacent nucleosomes with H3K36me3, a histone mark known to be involved in mediating HDR. To this end, I have engineered a Cas9- PRDM9 fusion construct. PRDM9 is a methyltransferase that deposits H3K4me3 and H3K36me3 to mark recombination hot spots during meiosis and has also been shown to act as a pioneer factor making chromatin more accessible for downstream processes. In preliminary experiments, this novel fusion construct displays a 2-fold improvement in HDR:indel ratio compared to Cas9 alone. By directly altering chromatin architecture, I hypothesize that HDR levels can be increased for precise genome editing and targeted DNA insertions across different cell types and regardless of preexisting chromatin architecture. In the first aim, I plan to investigate how site-specific chromatin modifications mediate DNA repair following cutting by CRISPR-Cas9. I will also study whether direct reconfiguration of chromatin architecture via Cas9-PRDM9 fusion can improve homologous directed repair (HDR). In the second aim, I will investigate the effect on HDR of blunt vs staggered cuts introduced by different Cas12 family nucleases, including a newly identified hypercompact CasΦ, and whether fusions of these editors with PRDM9 can improve precise genome editing. Finally, I will investigate how these combined properties can be harnessed to improve large DNA insertions (>100bp) in both cell lines and human primary T cells.