The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells

Marina Mohr, Nkerorema Damas, Johanne Gudmand-Høyer, Katrine Zeeberg, Dominika Jedrzejczyk, Arsenios Vlassis, Martí Morera-Gómez, Sara Pereira-Schoning, Urška Puš, Anna Oliver-Almirall, Tanja Lyholm Jensen, Roland Baumgartner, Brian Tate Weinert, Ryan T. Gill*, Tanya Warnecke*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

CRISPR-Cas12a nucleases have expanded the toolbox for targeted genome engineering in a broad range of organisms. Here, using a high-throughput engineering approach, we explored the potential of a novel CRISPR-MAD7 system for genome editing in human cells. We evaluated several thousand optimization conditions and demonstrated accurate genome reprogramming with modified MAD7. We identified crRNAs that allow for ≤95% non-homologous end joining (NHEJ) and 66% frameshift mutations in various genes and observed the high-cleavage fidelity of MAD7 resulting in undetectable off-target activity. We explored the dsDNA delivery efficiency of CRISPR-MAD7, and by using our optimized transfection protocol, we obtained ≤85% chimeric antigen receptor (CAR) insertions in primary T cells, thus exceeding the baseline integration efficiencies of therapeutically relevant transgenes using currently available virus-free technologies. Finally, we evaluated multiplex editing efficiency with CRISPR-MAD7 and demonstrated simultaneous ≤35% CAR transgene insertions and ≤80% gene disruption efficiencies. Both the platform and our transfection procedure are easily adaptable for further preclinical studies and could potentially be used for clinical manufacturing of CAR T cells.
Original languageEnglish
JournalACS Synthetic Biology
Volume12
Issue number2
Pages (from-to)375-389
ISSN2161-5063
DOIs
Publication statusPublished - 2023

Keywords

  • CRISPR
  • MAD7
  • NHEJ
  • HDR
  • Frameshift mutations
  • CAR T-cells

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