Uncovering unintended modifications with an in-depth assessment using Xdrop™
As CRISPR-based technologies gain traction in sensitive applications of genome editing and engineering, verifying the presence of (un)intended modifications has become crucial. Standard PCR approaches for validation focus only on the immediate vicinity of the edit site. Xdrop™ is different.
Xdrop™ allows scrutinizing ~100 kb around the edit sites by enabling targeted sequencing of long DNA fragments. Automated and designed with an easy workflow that is accessible to any lab, Xdrop™ can reveal unintended modifications where standard PCR methods can’t.
Using Xdrop™, we enriched long fragments of DNA isolated from isogenic human cell lines CRISPR-engineered to differ at two single-nucleotide positions on Exon 4 of the APOE gene.
Focusing on two of those cell lines with (supposedly) homozygous haplotypes ε3/ε3 and ε2/ε2, we used Indirect Sequence Capture to sort out single molecules of interest based on the presence of a short sequence (Detection Sequence) ~2 kb upstream of the edit sites.
These molecules were then amplified by droplet MDA and prepared for sequencing on Oxford Nanopore (ONT) and Illumina (ILL) instruments. In both sequencing approaches, enrichment was roughly 200x for a 100 kb region and as high as ~1000x for a 10 kb region centered on the Detection Sequence and including the edit sites. Targeted enrichment was achieved using a single primer set and only 10 ng of input DNA.
See the Application Note below for a graphical representation of the sequencing depth.
Although the edit accuracy, integrity and haplotype of the two cell lines had been assessed by PCR and Sanger sequencing, we uncovered an unintended insertion of a co-transfection plasmid immediately downstream from the edit sites. The insert affected only one haplotype in both cell lines, expanding the primer distance for the edit assessment from 227 bp to >3.5 kb.
The expansion prevented amplification of the affected allele and therefore, the standard PCR-based assessment only detected the unaffected haplotype. Because both cell lines seemed homozygous as expected after editing, the failed amplification went unnoticed.
The Indirect Sequence Capture of long DNA molecules and the single-molecule amplification of Xdrop™ ensured the discovery of the unwanted insertion.