Fig. 2: Multiplexed immune cell gene editing with LbCas12a mice and enAsCas12a mice.
From: Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering
a, Schematic showing the ex vivo workflow for multiplexed gene editing in primary immune cells (of both LbCas12a mice and LSL-enAsCas12a mice). CD8+ T cells and BMDCs were isolated from the spleen and bone marrow of LbCas12a or LSL-enAsCas12a mice, followed by ex vivo culture and retroviral infection. FACS was used to analyse the efficiency of single-cell-level DKO and sort for infected cells for downstream molecular analysis. Created in BioRender. Chen, S. (2025) https://BioRender.com/d80g206. b, Quantification of CD24 or CD11c negative BMDCs percentage for the guides compared with the vector control. Unpaired two-sided t-test was used to assess significance. N = 15 technical replicates for all groups. c, Quantification showing the percentage of CD8a or Thy1 negative CD8+ T cells for the guides compared with the vector control. Unpaired two-sided t-test was used to assess significance. N = 3 technical replicates for CD8 groups, and N = 10 technical replicates for Thy1 groups. d, Quantification showing the percentage of Thy1, Cxcr4 and CD4 negative CD4+ T cells for the guides compared with the vector control. Unpaired two-sided t-test was used to assess significance. Technical replicates for all groups. N = 10 for Thy1 groups. N = 5 for Cxcr4 groups. N = 3 for CD4 groups. e, Flow cytometry analysis on the CD24-CD43 DKO experiment in LbCas12a BMDCs. Left: representative FACS plot demonstrating CD24 and CD43 expression in BMDCs. Right: quantification of CD24 and CD43 DKO efficiency in different groups. One-way ANOVA with Dunnett’s multiple comparisons test was used to assess significance. N = 3 biological replicates for all groups. f, Flow cytometry analysis on the CD24-CD11c DKO experiment in LbCas12a BMDCs. Left: representative FACS plot demonstrating CD24 and CD11c expression in BMDCs. Right: quantification of CD24 and CD11c DKO efficiency in different groups. One-way ANOVA with Dunnett’s multiple comparisons test was used to assess significance. N = 3 biological replicates for all groups. g, Schematic showing the retroviral vector design for LSL-enAsCas12a immune cell editing. crRNA array was expressed by human U6 promoter (hU6). Cre and mScarlet expression was driven by EFS promoter to induce enAsCas12a-HF1 expression and to label the infected cells, respectively. CD8+ T cells and BMDCs were isolated from the spleen and bone marrow of LSL-enAsCas12a mice, as shown in a. Partial element created in BioRender. Chen, S. (2025) https://BioRender.com/b54g776. h, Flow cytometry analysis on the CD24-CD11c DKO experiment in LSL-enAsCas12a BMDCs. Left: representative FACS plots demonstrating CD24 and CD11c expression in BMDCs. Right: quantification of CD24 and CD11c DKO efficiency in different groups. One-way ANOVA with Dunnett’s multiple comparisons test was used to assess significance. N = 4 biological replicates for all groups. i, Targeted sequencing quantifies percent gene modification for the CD24-CD11c DKO experiment in BMDCs. Two-way ANOVA with Dunnett’s multiple comparisons test was used to assess significance. For all groups, N = 3 biological replicates. j, Flow cytometry analysis on the CD80-H2Ab1 DKO experiment in LSL-enAsCas12a BMDCs. Left: representative FACS plots demonstrating CD80 and MHC II expression in BMDCs. Right: quantification of CD80 and MHC II DKO efficiency in different groups. One-way ANOVA with Dunnett’s multiple comparisons test was used to assess significance. N = 3 biological replicates for all groups. k, Targeted sequencing quantifies percent gene modification for the CD80-H2Ab1 DKO experiment in BMDCs. Two-way ANOVA with Dunnett’s multiple comparisons test was used to assess significance. For all groups, N = 3 biological replicates. l, Flow cytometry analysis on the Thy1-CD27 DKO experiment in LSL-enAsCas12a CD8+ T cells. Left: representative FACS plots demonstrating CD27 and Thy1 expression in CD8+ T cells. Right: quantification of CD27 and Thy1 DKO efficiency in different groups. One-way ANOVA with Dunnett’s multiple comparisons test was used to assess significance. N = 3 biological replicates for all groups. m, Flow cytometry analysis comparing DKO efficiency in BMDCs of LSL-LbCas12a and LSL-enAsCas12a mice. Left: representative FACS plots demonstrating CD24 and CD11c expression in BMDCs. Right: quantification of CD24 and CD11c DKO efficiency in different groups. One-way ANOVA with Tukey’s multiple comparisons test was used to assess significance. N = 4 technical replicates for all groups. All bar plots shown as mean ± s.e.m. Exact P values are labelled.
