Fig. 6: CAR-T engineering by enGager with superior efficiency than WT Cas9.

A Schematic diagram of Knock in strategy of 3Kb CD19/CD22 dual CAR (css62) cssDNA donor templates for TRAC locus in primary T cells. B Quantification of % of CD19/CD22 CAR Knock-in using cssDNA donor analyzed by CD19 binder for various doses of Cas9 WT and Cas9-GS-FECO enGager mRNA at 1 ug, 2 ug and 4 ug. Data were collected at day 7 and day 11 post electroporation of primary T cells. Cas9-GS-FECO enGager achieves ~ 4- to 6-fold higher CAR-T engineering efficiency than Cas9 WT. Bars represents mean ± SD from 2 biological replicates. C Characterization of the engineered CAR-T cells or mock treated T cells for total cell count, cell proliferation fold change and cell viability over time. D NALM6 leukemia lymphocyte killing curve of unengineered T cells, CD19-CD22 dual CAR-T cells engineered with 2 ug of WT Cas9 mRNA and 2 ug of GS-FECO enGager mRNA over the course of 96 h. Effect (T cells): Target (NALM6 cells) are at 2.25:1 for left panel, 4.5:1 for middle panel and 9:1 for right panel. E NALM6 cell killing function of CAR-T cells at 24 h for E:T ratio at 2.25:1, 4.5:1 and 9:1. Bars represents mean ± SD from 2 biological replicates. F Schematic diagram of engineered enGagers with single stranded DNA binding protein (SSBP) can recruit cssDNA donor template and form a tripartite editing machinery for efficient translocation of the entire editing complex from cytoplasm to nucleus. As a result, the donor DNA has higher effective local concentration in the nucleus for more efficient homologous directed genome integration. This process works more prominently with cssDNA. Illustrations for panel (F) is generated with BioRender.