CRISPR-Cas12a-integrated transgenes in genomic safe-harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells. CRISPR-MAD7 Genomic Safe Harbor Sites

Discovery of genomic safe harbor sites (SHS) is fundamental for multiple transgene integrations, such as reporter genes, chimeric antigen receptors (CAR), and safety switches, which are required for safe cell products for regenerative cell therapies and immunotherapies. Here we identified and characterized potential SHS in human cells. Using the CRISPR-MAD7 system, we integrated transgenes at these sites in iPSC, primary T and NK cells, and Jurkat cell line, and demonstrated efficient and stable expression at these loci. Subsequently, we validated the differentiation potential of engineered iPSC towards CD34+ hematopoietic stem and progenitor cells (HSPC), lymphoid progenitors (LPC), and natural killer (NK) cells, and showed that transgene expression was perpetuated in these lineages. Finally, we demonstrated that engineered iPSC-derived NK cells retained expression of a non-virally integrated anti-CD19 CAR, suggesting that several of the investigated SHS can be used to engineer cells for adoptive immunotherapies.