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Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering
Thursday, 2025/04/03 | 08:20:06
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Kaiyuan Tang, Liqun Zhou, Xiaolong Tian, Shao-Yu Fang, Erica Vandenbulcke, Andrew Du, Johanna Shen, Hanbing Cao, Jerry Zhou, Krista Chen, Hyunu R. Kim, Zhicheng Luo, Shan Xin, Shawn H. Lin, Daniel Park, Luojia Yang, Yueqi Zhang, Kazushi Suzuki, Medha Majety, Xinyu Ling, Stanley Z. Lam, Ryan D. Chow, Ping Ren, Bo Tao, Keyi Li, Adan Codina, Xiaoyun Dai, Xingbo Shang, Suxia Bai, Timothy Nottoli, Andre Levchenko, Carmen J. Booth, Chen Liu, Rong Fan, Matthew B. Dong, Xiaoyu Zhou & Sidi Chen
Nature Biomedical and Engineering (2025); Published online: 20 March 2025
AbstractThe pleiotropic effects of human disease and the complex nature of gene-interaction networks require knock-in mice allowing for multiplexed gene perturbations. Here we describe a series of knock-in mice with a C57BL/6 background and with the conditional or constitutive expression of LbCas12a or of high-fidelity enhanced AsCas12a, which were inserted at the Rosa26 locus. The constitutive expression of Cas12a in the mice did not lead to discernible pathology and enabled efficient multiplexed genome engineering. We used the mice for the retrovirus-based immune-cell engineering of CD4+ and CD8+ T cells, B cells and bone-marrow-derived dendritic cells, for autochthonous cancer modelling through the delivery of multiple CRISPR RNAs as a single array using adeno-associated viruses, and for the targeted genome editing of liver tissue using lipid nanoparticles. We also describe a system for simultaneous dual-gene activation and knockout (DAKO). The Cas12a-knock-in mice and the viral and non-viral delivery vehicles provide a versatile toolkit for ex vivo and in vivo applications in genome editing, disease modelling and immune-cell engineering, and for the deconvolution of complex gene interactions.
See https://www.nature.com/articles/s41551-025-01371-2
Figure 1 Generation of conditional and constitutive LbCas12a and enAsCas12a knock-in mice. 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.
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