A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes

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Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.

Original languageEnglish (US)
Article number9749
JournalInternational journal of molecular sciences
Issue number17
StatePublished - Sep 2022

Bibliographical note

Funding Information:
This study makes use of data generated by the Blueprint Consortium (see A). A full list of the investigators who contributed to the generation of the data is available from www.blueprint-epigenome.eu , accessed on 10 October 2021) Funding for the project was provided by the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no 282510–BLUEPRINT. All illustrations were made with Biorender.com , accessed on 30 July 2022.

Publisher Copyright:
© 2022 by the authors.


  • CRISPR-Cas9
  • base editor
  • genome engineering
  • pan-RNase inhibitor
  • primary human monocytes


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