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Light-activated cell identification and sorting (LACIS) for selection of edited clones on a nanofluidic device.

  • Author(s): Mocciaro, Annamaria;
  • Roth, Theodore L;
  • Bennett, Hayley M;
  • Soumillon, Magali;
  • Shah, Abhik;
  • Hiatt, Joseph;
  • Chapman, Kevin;
  • Marson, Alexander;
  • Lavieu, Gregory
  • et al.
Abstract

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.

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