Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells — ASN Events
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  2. Poster Session 2
  3. Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells

Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells (#203)

Hyuncheol Jung 1 2 3 , Pascal Devant 1 , Carter Ching 3 , Mineta Ota 1 , Emma Dann 1 4 5 , Ronghui Zhu 1 4 5 , Chandrima Modak 1 4 5 , Ana Vasquez-Ibarra 1 , Jennifer R Hamilton 6 7 , Zachary Steinhart 1 2 , Wayne Ngo 7 8 9 , Luis Sandoval 3 , Jae Hyung Jung 3 , Jae Hyun J Lee 1 10 , Da Xu 6 , Meirui An 11 12 13 14 , Esha Urs 3 , Peixin Amy Chen 3 , Vincent Allain 1 3 15 , Takuya Tada 16 17 , Luke A Gilbert 18 19 20 , Brian Shy 1 21 22 , Jonathan K Pritchard 4 5 23 , James K Nuñez 6 24 , Nathaniel R Landau 16 17 , David R Liu 12 13 14 25 , Justin Eyquem 1 2 3 19 21 26 27 , Jennifer Doudna 1 6 7 8 9 28 29 30 , Jack Reid 3 , Alexander Marson 1 2 3 7 18 19 21 27 , Julia Carnevale 1 2 3 19 21
  1. Gladstone, UCSF Institute for Genomic Immunology, San Francisco, CA
  2. Parker Institute for Cancer Immunotherapy, San Francisco, CA
  3. Department of Medicine, University of California: San Francisco, San Francisco, CA, USA
  4. Department of Genetics, Stanford University, Stanford, CA
  5. Department of Biology, Stanford University, Stanford, CA
  6. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA
  7. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA
  8. Gladstone Institute for Data Science and Biotechnology, San Francisco, CA
  9. California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA
  10. Department of Laboratory Medicine, University of California: San Francisco, San Francisco, CA
  11. Broad Institute of MIT and Harvard, Cambridge
  12. Merkin Institute, Broad Institute of MIT and Harvard, Cambridge, MA
  13. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA
  14. Howard Hughes Medical Institute, Harvard University, Cambridge, MA
  15. Université Paris Cité, INSERM UMR1342, Hôpital Saint-Louis, Paris, France
  16. NYU Grossman School of Medicine, New York
  17. Department of Microbiology, NYU Grossman School of Medicine, New York, NY
  18. Arc Institute, Palo Alto
  19. UCSF Helen Diller Family Comprehensive Cancer Center, University of California: San Francisco, San Francisco, CA
  20. Department of Urology, University of California: San Francisco, San Francisco, CA
  21. Weill Cancer Hub West, Stanford University and UCSF, Stanford and San Francisco, CA
  22. Department of Laboratory Medicine, University of California: San Francisco, San Francisco, CA
  23. Stanford University, Stanford
  24. Chan Zuckerberg Biohub San Francisco, San Francisco, CA
  25. Harvard University, Cambridge
  26. Institute for Human Genetics, University of California: San Francisco, San Francisco, CA
  27. Department of Microbiology and Immunology, University of California: San Francisco, San Francisco, CA
  28. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA
  29. Lawrence Berkeley National Laboratory, Berkeley, CA
  30. Department of Chemistry, University of California, Berkeley, Berkeley, CA

Primary human myeloid cells are promising candidates for immunotherapy, yet efficient and scalable technologies for genetic engineering and screening in these cells remain limited.  We found that the previously described CRISPR gene-editing method for human myeloid cells, which relies on electroporation, caused substantial cell loss and rendered the surviving cells functionally inert. Our lab has developed a virus-like particle (VLP)-based toolkit that delivers diverse CRISPR genome-editing modalities to human monocytes, macrophages, and dendritic cells with high efficiency, while preserving cell viability and innate immune responsiveness. VLP-mediated delivery of ribonucleoprotein payloads supports gene knockout, base editing, and epigenetic silencing. Furthermore, when combined with AAV-mediated donor delivery, this approach enables site-specific integration of large DNA sequences via homology-directed repair. We also developed SLICeVLP, a system combining sgRNA delivery via VPX-lentivirus with Cas9 protein delivery via engineered virus-like particles (eVLPs), and applied it to perform pooled loss-of-function screens and Perturb-seq in primary human macrophages. These methods have shown broad applicability to primary human dendritic cells as well, which will enable pooled loss- or gain-of-function screens in these cells in the future. Additionally, our efforts to engineer eVLPs with scFv-based binders to confer tropism to different myeloid cell subsets may allow us to utilize the eVLP system for in-vivo particle delivery, further expanding the discovery power and translational relevance of our tool set. Taken together, this platform enables unbiased functional genomics discovery in primary human myeloid cells, with direct implications for myeloid cell therapy design. We plan to use this technology to uncover genetic modulations that enhance dendritic cell function—such as cross-presentation and priming of the adaptive immune system—to improve anti-tumoral responses.