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To isolate human cell clones with intended genotypes induced by genome editing, a large number of clones must be isolated by single-cell cloning. Selection markers such as drug-resistant genes can be used to enhance the chance of isolation of target cells, but they can also cause side effects by leaving unwanted modifications in the genome. Without using selection markers, laboratories typically perform limiting dilutions of cell suspensions, which is costly and time-consuming when CRISPR-Cas9CRISPR-Cas9 is inefficient at generating the intended genetic modifications. Therefore, we developed this protocol, with which more than 80 clones of genome-edited cultured human cells can be obtained in a one-time experiment by sorting EGFP-positive genome-edited cells with a microfluidic cell sorter and subsequently seeding single-cells using an automated cell dispensing system. Here we describe a method for introducing the c.2333G > T (p.R778L) point mutation in the ATP7B gene that causes Wilson’s disease into HeLa cells and establishing clones as an example.

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Efficient Single-Cell Cloning of Genome-Edited Cultured Human Cells

  • 实验方案

  • 发表日期：

    2025年

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  • Gou Takahashi,
  • Yuichiro Miyaoka

摘要

To isolate human cell clones with intended genotypes induced by genome editing, a large number of clones must be isolated by single-cell cloning. Selection markers such as drug-resistant genes can be used to enhance the chance of isolation of target cells, but they can also cause side effects by leaving unwanted modifications in the genome. Without using selection markers, laboratories typically perform limiting dilutions of cell suspensions, which is costly and time-consuming when CRISPR-Cas9CRISPR-Cas9 is inefficient at generating the intended genetic modifications. Therefore, we developed this protocol, with which more than 80 clones of genome-edited cultured human cells can be obtained in a one-time experiment by sorting EGFP-positive genome-edited cells with a microfluidic cell sorter and subsequently seeding single-cells using an automated cell dispensing system. Here we describe a method for introducing the c.2333G > T (p.R778L) point mutation in the ATP7B gene that causes Wilson’s disease into HeLa cells and establishing clones as an example.