Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells

  • Nat Protoc. 2025 Oct;20(10):2735-2770. doi: 10.1038/s41596-025-01154-8.
Srishti U Sahu  #  1  2  3 ,  Madalena Castro  #  1  2  3 ,  Joseph J Muldoon  4  5 ,  Kunica Asija  1  2  3 ,  Stacia K Wyman  1 ,  Netravathi Krishnappa  1 ,  Lorena de Oñate  1  2 ,  Justin Eyquem  4  5  6  7  8  9 ,  David N Nguyen  1  4  5 ,  Ross C Wilson  10  11  12
Affiliations
  • 1. Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA.
  • 2. Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA.
  • 3. California Institute for Quantitative Biosciences at University of California Berkeley, Berkeley, CA, USA.
  • 4. Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
  • 5. Department of Medicine, University of California San Francisco, San Francisco, CA, USA.
  • 6. Parker Institute for Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA.
  • 7. Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA, USA.
  • 8. UCSF Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA.
  • 9. Institute for Human Genetics, University of California San Francisco, San Francisco, CA, USA.
  • 10. Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA. [email protected].
  • 11. Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA. [email protected].
  • 12. California Institute for Quantitative Biosciences at University of California Berkeley, Berkeley, CA, USA. [email protected].
  • # Contributed equally.
PMID: 40032999 DOI: 10.1038/s41596-025-01154-8
Abstract

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein Enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in and base editing. The protocol involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) homology-directed repair template (HDRT) DNA may be included. In contrast to electroporation, PERC is appealing because it needs no dedicated hardware and has less impact on cell phenotype and viability. Because of the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Editing efficiencies can surpass 90% when using either Cas9 or Cas12a in primary T cells or HSPCs. After 3 h dedicated to reagent preparation, the PERC delivery step can be completed in 1 h, with the associated Cell Culture steps taking 3-7 d total. Because the protocol calls for only three readily available reagents (protein, RNA and peptide) and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes the protocol appealing for rapid deployment in research and clinical settings.

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