Key Points Genomic disruption of CD7 prior to CAR transduction allows generation of CD7 CAR T cells without extensive self-antigen-driven fratricide. CD7 CAR T cells have robust activity against T-cell malignancies in vitro and in vivo.

SUMMARY Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time-consuming and expensive. Here we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs, or Cas9-guide-RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (~75%). These techniques enabled rapid evaluation of the functional effects of gene loss of Eed, Suz12, and DNMT3A. We also achieved homology-directed repair in primary human HSPCs (>20%). These methods will significantly expand applications for CRISPR/Cas9 technologies for studying normal and malignant hematopoiesis.