Federal Research Center Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Curr Protoc Hum Genet. 2020 Jun;106(1):e100. doi: 10.1002/cphg.100.
In neurodegeneration studies, researchers are faced with problems such as limited material availability and late disease manifestation. Cell models provide the opportunity to investigate molecular mechanisms of pathogenesis. Moreover, genome editing technologies enable generation of isogenic cell models of hereditary diseases. Our protocol outlines an approach for introducing an expanded CAG repeat tract into the first exon of the HTT gene, the Huntington's disease causing mutation. The protocol allows modeling the disease at various severity levels by introducing different numbers of CAG repeats. Furthermore, the protocol can be applicable for modeling other diseases caused by trinucleotide repeat expansion. It is important to note there are many difficulties with cloning repeated sequences and amplification of GC-rich regions. Here, we also propose troubleshooting options, which overcome these problems. The protocol is based on CRISPR/Cas9-mediated homologous recombination with a uniquely designed donor plasmid harboring an expanded CAG tract flanked with long homology arms. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Design and assembling donor and CRISPR/Cas9-expressing plasmids Basic Protocol 2: Transfection of cells with plasmids and sorting GFP-positive cells Basic Protocol 3: PCR screening single-cell clones and validation of the mutant HTT expression.
在神经退行性疾病研究中，研究人员面临着材料有限和疾病晚期表现等问题。细胞模型为研究发病机制的分子机制提供了机会。此外，基因组编辑技术可用于产生遗传性疾病的同源细胞模型。本方案概述了一种将扩展的 CAG 重复序列引入 HTT 基因（亨廷顿病致病突变）第一外显子的方法。该方案可通过引入不同数量的 CAG 重复来模拟不同严重程度的疾病。此外，该方案可适用于模拟其他由三核苷酸重复扩展引起的疾病。需要注意的是，克隆重复序列和扩增 GC 丰富区域存在许多困难。在这里，我们还提出了故障排除选项，以克服这些问题。该方案基于 CRISPR/Cas9 介导的同源重组，使用独特设计的携带扩展 CAG 序列的供体质粒，两侧带有长同源臂。© 2020 Wiley Periodicals LLC. 基本方案 1：设计和组装供体和 CRISPR/Cas9 表达质粒 基本方案 2：用质粒转染细胞和分选 GFP 阳性细胞 基本方案 3：PCR 筛选单细胞克隆和验证突变 HTT 表达。
