Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering

Cell Rep. 2021 Jun 1;35(9):109207. doi: 10.1016/j.celrep.2021.109207.


As genome engineering advances cell-based therapies, a versatile approach to introducing both CRISPR-Cas9 ribonucleoproteins (RNPs) and therapeutic transgenes into specific cells would be transformative. Autologous T cells expressing a chimeric antigen receptor (CAR) manufactured by viral transduction are approved to treat multiple blood cancers, but additional genetic modifications to alter cell programs will likely be required to treat solid tumors and for allogeneic cellular therapies. We have developed a one-step strategy using engineered lentiviral particles to introduce Cas9 RNPs and a CAR transgene into primary human T cells without electroporation. Furthermore, programming particle tropism allows us to target a specific cell type within a mixed cell population. As a proof-of-concept, we show that HIV-1 envelope targeted particles to edit CD4+ cells while sparing co-cultured CD8+ cells. This adaptable approach to immune cell engineering ex vivo provides a strategy applicable to the genetic modification of targeted somatic cells in vivo.

Keywords: CAR-T cells; CRISPR delivery; CRISPR-Cas9; precision genome editing; viral engineering; virus-like particles.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • A549 Cells
  • CD4-Positive T-Lymphocytes / metabolism
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems / genetics*
  • Cell Engineering*
  • Gene Editing
  • Gene Transfer Techniques*
  • HIV-1 / physiology
  • Humans
  • Jurkat Cells
  • Lentivirus / genetics
  • Receptors, Chimeric Antigen / metabolism
  • Ribonucleoproteins / metabolism
  • Transgenes*
  • Virion / metabolism
  • env Gene Products, Human Immunodeficiency Virus


  • Receptors, Chimeric Antigen
  • Ribonucleoproteins
  • env Gene Products, Human Immunodeficiency Virus
  • CRISPR-Associated Protein 9