PINE-TREE可实现对人类细胞系的高效基因改造。
PINE-TREE enables highly efficient genetic modification of human cell lines.
作者信息
Frisch Carlye, Kostes William W, Galyon Brooke, Whitman Brycelyn, Tekel Stefan J, Standage-Beier Kylie, Srinivasan Gayathri, Wang Xiao, Brafman David A
机构信息
School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85287, USA.
Molecular and Cellular Biology Graduate Program, Arizona State University, Tempe, AZ 85287, USA.
出版信息
Mol Ther Nucleic Acids. 2023 Jul 17;33:483-492. doi: 10.1016/j.omtn.2023.07.007. eCollection 2023 Sep 12.
Prime editing technologies enable precise genome editing without the caveats of CRISPR nuclease-based methods. Nonetheless, current approaches to identify and isolate prime-edited cell populations are inefficient. Here, we established a fluorescence-based system, prime-induced nucleotide engineering using a transient reporter for editing enrichment (PINE-TREE), for real-time enrichment of prime-edited cell populations. We demonstrated the broad utility of PINE-TREE for highly efficient introduction of substitutions, insertions, and deletions at various genomic loci. Finally, we employ PINE-TREE to rapidly and efficiently generate clonal isogenic human pluripotent stem cell lines, a cell type recalcitrant to genome editing.
碱基编辑技术能够实现精确的基因组编辑，而不存在基于CRISPR核酸酶方法的那些限制条件。尽管如此，目前用于鉴定和分离碱基编辑细胞群体的方法效率较低。在此，我们建立了一种基于荧光的系统——利用瞬时报告基因进行编辑富集的碱基诱导核苷酸工程（PINE-TREE），用于实时富集碱基编辑细胞群体。我们证明了PINE-TREE在多个基因组位点高效引入替换、插入和缺失方面具有广泛的实用性。最后，我们利用PINE-TREE快速高效地生成了克隆同基因人类多能干细胞系，这是一种对基因组编辑具有抗性的细胞类型。
Zotero 插件