Title: Correction of autoimmune IL2RA mutations in primary human T cells u sing non - viral genome targeting

The full promise of cell-based immunotherapies depends on technology to engineer and correct targeted genome sequences in primary human immune cells. CRISPR-Cas9 genome editing components can be electroporated into primary cells for gene knock-out. To date, co-delivery of oligodeoxynucleotide homology-directed repair (HDR) templates has enabled the replacement of short stretches of nucleotides; however efficient delivery of longer HDR templates has required viral-encoded templates, limiting adaptability and therapeutic applications. Here, we describe methods for non-viral T cell genome targeting with Cas9 RNPs and long (>1 kilobase) non-viral HDR templates. Targeting was efficient across multiple blood donors and genomic loci, cell viability was high, and the procedure could be multiplexed for bi-allelic or multi-gene targeting. Long single-stranded (ss)DNA HDR templates limited observed off-target integrations using either Cas9 or a Cas9 nickase. We were able to identify the causal mutations in IL2RA (interleukin-2 receptor alpha; CD25) in multiple siblings with monogenic autoimmunity and correct the mutations in their affected primary T cells. Non-viral genome targeting will allow rapid and flexible experimental manipulation of primary human immune cells and therapeutic engineering of patient cells.