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Generation of cell lines with specific mutations is integral to the in vitro study of many diseases and the associated pathogenesis, and the CRISPR-Cas9 gene editing system has revolutionized the ability to efficiently generate disease models. Limiting dilution and FACS have traditionally been used to obtain clonal cell lines with specific genomic modifications introduced by the CRISPR-Cas9 system; however, they require large sample sizes and often yield low cell viability. The authors used the CellRaft® Technology to sort cells based on the temporal evolution of fluorescent protein expression (EGFP) to generate a CRISPR gene-edited cell line with a leukemia-associated mutation (S34F) in the U2AF1 protein, allowing for the further study of the consequences of this mutation on mRNA splicing in AML.
CRISPR nucleases can introduce damages to DNA strands that when repaired by the cell, result in introduction or deletion of nucleotides to the sequence. Presented here is a strategy that utilizes a ‘sequence-ascertained favorable editing’ (SAFE) donor approach that has the ability to make such edits to the sequence of DNA using combinations of substitutions unlikely to have any effect on the target site. To test and evaluate the success of the SAFE donor editing approach, two target sites in the genes TEX2 and TTF1 were chosen as these are known to display frequent deletions affecting the editing site. Human stem cells were electroporated, seeded in 12-well plates to recover before being passaged for use with the CellRaft Technology. Cells were seeded on a CellRaft Array and scanned for 5 days before being isolated into 96-well collection plates. A total of 437 and 435 clones from each target site TEX2 and TTF1 pools, respectively were isolated and evaluated downstream. DNA was extracted from the clones, amplified, and the sequences were analyzed. The use of the SAFE donor approach easily enabled the detection of unintended effects by sequencing the target site.