使用新方法挑選對基因編輯過的克隆

論文標題：Light-activated cell identification and sorting (LACIS) for selection of edited clones on a nanofluidic device

作者：Annamaria Mocciaro, Theodore L. Roth, Hayley M. Bennett, Magali Soumillon, Abhik Shah, Joseph Hiatt, Kevin Chapman, Alexander Marson, Gregory Lavieu

數字識別碼：10.1038/s42003-018-0034-6

儘管CRISPR分子工具箱有所改進，鑒定和純化正確編輯的細胞克隆仍然效率低下，費力且低產。近日，《通訊-生物學》發表的一項研究Light-activated cell identification and sorting (LACIS) for selection of edited clones on a nanofluidic device提出了一種新的方法。

加州大學舊金山分校的Alexander Marson、Berkeley Lights的Gregory Lavieu及其同事發明的新方法使用光電定位技術在納米流體設備上進行單細胞操作，從而實現對正確編輯的細胞克隆的分離、分選和擴增。簡單來說，就是使用以CXCR4為靶細胞的Cas9核糖核蛋白對原代T細胞進行電穿孔，進而實現在晶元上將單個T細胞分離並使其擴增成集落。他們通過CXCR4細胞表面染色在晶元上快速進行編輯表型結果評估。此外，細胞集落會根據基因型進一步被識別區分。細胞集落會被依次分離，從晶元上轉移下來單獨進行靶標測序，細胞克隆測序結果得到驗證後會進行晶元外擴增。

這種方法能夠在Cas9核糖核蛋白引入細胞後10天之內評估單個克隆基因編輯效率，包括單、雙等位基因插入/缺失或精確核苷酸替換的速度。

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圖1：高精度識別和分選基因編輯細胞的方法

摘要：Despite improvements in the CRISPR molecular toolbox, identifying and purifying properly edited clones remains slow, laborious, and low-yield. Here, we establish a method to enable clonal isolation, selection, and expansion of properly edited cells, using OptoElectroPositioning technology for single-cell manipulation on a nanofluidic device. Briefly, after electroporation of primary T cells with CXCR4-targeting Cas9 ribonucleoproteins, single T cells are isolated on a chip and expanded into colonies. Phenotypic consequences of editing are rapidly assessed on-chip with cell-surface staining for CXCR4. Furthermore, individual colonies are identified based on their specific genotype. Each colony is split and sequentially exported for on-target sequencing and further off-chip clonal expansion of the validated clones. Using this method, single-clone editing efficiencies, including the rate of mono- and bi-allelic indels or precise nucleotide replacements, can be assessed within 10 days from Cas9 ribonucleoprotein introduction in cells.

閱讀論文全文請訪問：http://t.cn/EyKPb8S

期刊介紹：Communications Biology(https://www.nature.com/commsbio/) is an open access journal from Nature Research publishing high-quality research, reviews and commentary in all areas of the biological sciences. Research papers published by the journal represent significant advances bringing new biological insight to a specialized area of research.

（來源：Communications Biology）

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