![[feed]](/style/images/feed-icon-14x14.png){kind=icon}
Tools
Tools
Preview |
PDF (Original Article)
- Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
7MB |
![[thumbnail of Supplemental Information]](https://edoc.mdc-berlin.de/style/images/fileicons/other.png) |
Other (Supplemental Information)
2MB |
| Item Type: | Article |
|---|---|
| Title: | Single-stranded HDR templates with truncated Cas12a-binding sequences improve knock-in efficiencies in primary human T cells |
| Creators Name: | Nitulescu, A.M., Du, W., Glaser, V., Kath, J., Aird, E.J., Cullot, G., Greensmith, R., Mikkelsen, N.S., Stein, M., Bak, R.O., Kaminski, M., Corn, J.E. and Wagner, D.L. |
| Abstract: | CRISPR-Cas12a gene editing 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 donor-based editing is subject to various factors, with template format playing a crucial role. Currently, the predominant non-viral template format for homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA, which is toxic when transfected at high doses. Others have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a 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. Additionally, we rule out ssDNase activity of AsCas12a under cell-physiological Mg2(+) conditions. Finally, we showcase the advantage of ssDNA donors with CTS (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three TCR-CD3 complex genes (TRAC, CD3ζ, CD3ε), achieving up to 90% knock-in rates for a 0.8kb-insert at the CD3ε locus. Long-read sequencing confirmed higher HDR rates and revealed that CTS reduced partial integration events compared to unmodified ssDNA. Overall, AsCas12a and ssCTS represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity. |
| Keywords: | MT, RNA/DNA Editing, Gene Editing, CRISPR-Cas12a, Non-Viral, Electroporation, SsDNA, HDR, CAR-T Cells, Cas-Target-Sequences, AsCas12a |
| Source: | Molecular Therapy Nucleic Acids |
| ISSN: | 2162-2531 |
| Publisher: | Cell Press / Elsevier |
| Volume: | 36 |
| Number: | 2 |
| Page Range: | 102568 |
| Number of Pages: | 1 |
| Date: | 10 June 2025 |
| Official Publication: | https://doi.org/10.1016/j.omtn.2025.102568 |
Repository Staff Only: item control page
