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Improving non-viral knock-in with modified single-stranded DNAs and small molecules

Nature Biotechnology volume 41, pages 478–479 (2023)Cite this article

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The addition of Cas9 target sequences to long single-stranded DNAs, combined with cocktails of small molecules to boost homology-directed repair, leads to marked enhancement of non-viral knock-in efficiency and yield in primary human T cells and other hematopoietic cell types.

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Fig. 1: ssDNA CTS templates and small molecules improve non-viral knock-in yield and purity.

References

  1. Roth, T. L. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405–409 (2018). This paper describes a non-viral knock-in strategy with naked DNA and reports lower toxicity and off-target integration with ssDNA templates.

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  2. Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017). This paper describes an AAV-mediated knock-in strategy for CAR-T cell generation.

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  3. Nguyen, D. N. & Roth, T. L. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nat. Biotechnol. 38, 44–49 (2020). This paper describes enhancement of knock-in efficiency through inclusion of Cas9 target sequences on dsDNA templates.

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  4. Lin-Shiao, E. et al. CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells. Nucleic Acids Res. 50, 1256–1268 (2022). This paper shows a variety of DNA origami structured CTS templates.

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This is a summary of: Shy, B. R. et al. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat. Biotechnol. https://doi.org/10.1038/s41587-022-01418-8 (2022).

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Improving non-viral knock-in with modified single-stranded DNAs and small molecules. Nat Biotechnol 41, 478–479 (2023). https://doi.org/10.1038/s41587-022-01420-0

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