Verification of CRISPR editing by Xdrop™ Indirect Sequence Capture followed by short- and long- read sequencing

Validation of CRISPR-Cas9 editing typically explore the immediate vicinity of the target site and distal off-target sequences, which have led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kb and only affect one allele.

The golden standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach will allow detection of smaller editing events but will fail to detect larger rearrangements in particular when these only affect one of the two alleles. Detection of larger rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing.

Here we present a new microfluidic technology Xdrop™ that allows targeted enrichment of longer regions (~ 100 kb) using just a standard PCR primer set. We sequenced the enriched region on long- and short- read sequencing platforms to unravel unknown and unintended genome edits resulting from CRISPR-Cas9 gene editing.

We show how loss of heterozygosity, due to an accidental insertion in one of the alleles, remained undetected using standard procedures but was discovered implementing the Xdrop™ system. This demonstrates the potential of such technology in CRISPR gene editing assessments, providing an extremely accurate tool.