biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at the ACTB locus

• 全文链接

CRISPR/ cas9介导的大功能DNA片段在ACTB位点的高效对接

关键词：

来源：

Synthetic Biology

全文链接：

//agri.nais.net.cn/topic/downloadFile/c92f5001-8579-4b53-8f8e-457a524d2fcc

来源地址：

https://academic.oup.com/synbio/article/10/1/ysaf003/8010902?searchresult=1

资源所属：

马铃薯基因组设计育种

类型：

学术文献

语种：

英语

原文发布日期：

2025-02-13

摘要：

Recent advances in molecular and cell biology and imaging have unprecedentedly enabled multiscale structure-functional studies of entire metabolic pathways from atomic to micrometer resolution and the visualization of macromolecular complexes in situ, especially if these molecules are expressed with appropriately engineered and easily detectable tags. However, genome editing in eukaryotic cells is challenging when generating stable cell lines loaded with large DNA cargoes. To address this limitation, here, we have conceived biGMamAct, a system that allows the straightforward assembly of a multitude of genetic modules and their subsequent integration in the genome at the ACTB locus with high efficacy, through standardized cloning steps. Our system comprises a set of modular plasmids for mammalian expression, which can be efficiently docked into the genome in tandem with a validated Cas9/sgRNA pair through homologous-independent targeted insertion. As a proof of concept, we have generated a stable cell line loaded with an 18.3-kilobase-long DNA cargo to express six fluorescently tagged proteins and simultaneously visualize five different subcellular compartments. Our protocol leads from the in silico design to the genetic and functional characterization of single clones within 6 weeks and can be implemented by any researcher with familiarity with molecular biology and access to mammalian cell culturing infrastructure.