Publication: Detection of unintended on-target effects in CRISPR genome editing by DNA donors carrying diagnostic substitutions

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.

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