This protocol describes the isolation of single-cell clones from a CRISPR-edited pool and the expansion and genotyping of the subsequent clonal populations.

CRISPR-Cas9 can be used to generate knockout and knock-in cell pools that contain a heterogeneous mix of cells. Isolation and clonal expansion of individual cells from CRISPR-edited pools are often necessary to produce a monoclonal cell line that uniformly contains a single CRISPR edit at the target locus.

This protocol describes a limiting dilution technique using the standard cell culture method. We provide a detailed explanation of the following steps to isolate, expand, genotype, and store clonal populations from edited iPS cell pools:

  • Pool recovery and preparation for cloning
  • Limiting dilution
  • Clonal Expansion
  • Genotyping clones
  • Storage and additional