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

Nitulescu, A.-M.; Du, W.; Glaser, V.; Kath, J.; Greensmith, R.; Mikkelsen, N. S.; Stein, M.; Bak, R.; Kaminski, M.; Wagner, D. L.

doi:10.1101/2024.09.11.608426

bioRxiv · 10.1101/2024.09.11.608426

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

Nitulescu, A.-M.·Du, W.·Glaser, V.·Kath, J.·Greensmith, R.·Mikkelsen, N. S.·Stein, M.·Bak, R.·Kaminski, M.·Wagner, D. L.

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Abstract

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 template 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 mitigate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for AsCas12a 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 double-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, CD3{zeta}, CD3{varepsilon}), achieving up to 90% knock-in rates for a 0.8kb insert at the CD3{varepsilon} locus. Overall, AsCas12a Ultra and ssCTS donors represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.

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Nitulescu, A.-M., Du, W., Glaser, V., Kath, J., Greensmith, R., Mikkelsen, N. S., Stein, M., Bak, R., Kaminski, M., Wagner, D. L.. 2024-09-11. Single-stranded HDR templates with truncated Cas12a binding sequences improve knock-in efficiencies in primary human T cells. https://doi.org/10.1101/2024.09.11.608426

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