ArticleMolecular therapy. Nucleic acids2025
Single-stranded HDR templates with truncated Cas12a-binding sequences improve knock-in efficiencies in primary human T cells.
Article in Molecular therapy. Nucleic acids, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed.
- A de novo algorithm for allele reconstruction from Oxford nanopore amplicon reads, with application to CYP2D6.Bioinformatics (Oxford, England) · 2026Article
- Orthogonal CRISPR systems for targeted integration and multiplex base editing enable nonviral engineering of allogeneic CAR-T cells.Molecular therapy : the journal of the American Society of Gene Therapy · 2025Article
- Formulation of Recombinant Therapeutic Proteins: Technological Innovation, Regulations, and Evolution Towards Buffer-Free Formulations.Pharmaceutics · 2025Review
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13 authors.
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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 Mg
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