Characterizing and controlling CRISPR repair outcomes in nondividing human cells

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We showed that CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells. Neurons also took far longer to fully resolve this damage, and upregulated non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allowed us to direct DNA repair toward desired editing outcomes: in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing. Overall design: To determine how postmitotic neurons respond to Cas9-VLP treatment, we conducted bulk RNAseq on human iPSC-derived neurons (NGN2), before and after treatment with various types of VLPs and controls.