Single-stranded HDR templates with truncated Cas12a binding sequences improve knock-in efficiencies in primary human T cells

Non-viral gene editing via CRISPR-Cas12a offers an alternative to Cas9-based methods, providing better targeting of AT-rich regions, simplified guide RNA manufacturing, and high specificity. However, the efficacy of editing outcomes is subject to various factors, with tem-plate format playing a crucial role. Currently, the predominant non-viral template format for inducing homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA (dsDNA), which is toxic when transfected at high doses. Previous studies have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a repair template for Cas9-mediated gene editing can miti-gate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for As-Cas12a Ultra by exploring PAM orientation and binding requirements of the Cas12a-crRNA complex. Additionally, we rule out in vitro ssDNase activity of AsCas12a Ultra under cell-physiological Mg2+ conditions. Finally, we showcase the advantage of using ssDNA with dou-ble-stranded CTS end modifications (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three T-cell receptor-CD3 complex genes (TRAC, CD3z, CD3e), achieving up to 90% knock-in rates for a 0.8kb insert at the CD3e locus. Long-read sequencing of the on-target locus confirmed higher integration rates on a DNA level and re-vealed that double-stranded CTS end-modification reduced partial and reverse integration events compared to unmodified linear ssDNA. Overall, AsCas12a Ultra and ssCTS donors represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.