Light-Activated Cell Identification and Sorting (LACIS): A New Method to Identify and Select Edited Clones on a Microfluidic Device

Abstract

CRISPR-Cas9 gene editing has revolutionized cell engineering and promises to open new doors in gene and cell therapies. Despite improvements in the CRISPR-editing molecular toolbox in cell lines and primary cells, identifying and purifying properly edited clones remains slow, laborious and low-yield. Here, we establish a new method that uses cell manipulation on a chip with Opto-Electronic Positioning (OEP) technology to enable clonal isolation and selection of edited cells. We focused on editing CXCR4 in primary human T cells, a gene that encodes a co-receptor for HIV entry. T cells hold significant potential for cell-based therapy, but the gene-editing efficiency and expansion potential of these cells is limited. We describe here a method to obviate these limitations. Briefly, after electroporation of cells with CXCR4-targeting Cas9 ribonucleoproteins (RNPs), single T cells were isolated on a chip, where they proliferated over time into well-resolved colonies. Phenotypic consequences of genome editing could be rapidly assessed on-chip with cell-surface staining for CXCR4. Furthermore, independent of phenotype, individual colonies could be identified based on their specific genotype at the 5-10 cell stage. Each colony was split and sequentially exported for immediate on-target sequencing and validation, and further off-chip clonal expansion of the validated clones. We were able to assess single-clone editing efficiencies, including the rate of monoallelic and biallelic indels or precise nucleotide replacements. This new method will enable identification and selection of perfectly edited clones within 10 days from Cas9-RNP introduction in cells based on the phenotype and/or genotype.