Fig. 1: Generation of conditional and constitutive LbCas12a and enAsCas12a knock-in mice.
From: Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering
a, Schematic of the LSL-LbCas12a and LSL-enAsCas12a-HF1 Rosa26-targeting constructs for the two conditional transgenic lines. The backbone is Ai9 Rosa26-targeting vector. LbCas12a, labelled with HA tag and eGFP, is expressed under a CAG promoter. LoxP-(Stop)3XPolyA-LoxP (LSL) allows Cre-dependent conditional expression of LbCas12a protein. LSL-enAsCas12a mice follow the similar design, but are labeled with Myc Tag and use Egl-13 NLS instead of SV40 NLS on the N-terminus. Partial element created in BioRender. Chen, S. (2025) https://BioRender.com/y08x327. b, Generation of the constitutively active LbCas12a and enAsCas12a transgenic mouse line by crossing the corresponding conditional mouse line with the CMV-Cre mouse line. The LSL cassette was excised by Cre, leading to constitutive expression of LbCas12a or enAsCas12a protein. Partial element created in BioRender. Chen, S. (2025) https://BioRender.com/b54g776. c, Widefield fluorescence microscopy illustrating the expression of enAsCas12a-HF1-eGFP protein only in constitutive enAsCas12a mouse, but not in conditional LSL-enAsCas12a mouse or parental C57BL/6 mouse. d, Western blot showing the expression of enAsCas12a-HF1-MycTag protein in enAsCas12a mouse. enAsCas12a-HF1-MycTag protein was not detected in the protein lysate from LSL-enAsCas12a-HF1 and C57BL/6 mouse. Anti-MycTag antibody was used to detect enAsCas12a-HF1 protein and GAPDH was used as the internal control. e, IVIS spectrum imaging of GFP radiance from major organs in LSL-enAsCas12a mice, enAsCas12a-heterozygote mice and enAsCas12a-homozygote mice. Major organs include liver, spleen, kidney, heart, lung and brain. f, Quantification of IVIS GFP radiance. Two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test was used to assess significance. For all groups, N = 3 biological replicates. The dash line represents the average background expression from the LSL-enAsCas12a control mice (2.64 × 109). g, Left: white blood cell (WBC) count for constitutive enAsCas12a mice, LbCas12a mice and C57BL/6 control mice. Middle left: red blood cell (RBC) count for constitutive enAsCas12a mice, LbCas12a mice and C57BL/6 control mice. Middle right and right: comparison of lymphocytes/monocytes differential between constitutive enAsCas12a mice, LbCas12a mice and C57BL/6 control mice. One-way ANOVA with Tukey’s multiple comparisons test was used to assess significance. For bar plot, data are shown as mean ± s.e.m. For C57BL/6, N = 7 biological replicates. For enAsCas12a, N = 6 biological replicates. For LbCas12a, N = 6 biological replicates. Exact P values are labelled. h, Quantification of splenic immune cell percentage by flow cytometry. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess significance. All bar plots shown as mean ± s.e.m. Exact P values are labelled.
