Validate gene editing, including CRISPR edits

Reveal unintended on-target and off-target rearrangements using the Xdrop or Xdrop Sort workflows, which help avoid PCR bias

Xdrop and Xdrop Sort enable you to focus on target gene regions, supporting a range of downstream work, including long-read sequencing to:

  • Efficiently map CAR cassette integration

  • Characterize intended and unintended edits in engineered genes

  • Reveal unintended on-target and off-target mutations, deletions, or rearrangements

  • Identify insertions and gene duplications

Examples of these applications of Xdrop technology are given below.

Localizing lentivirus transduction-inserted CAR cassettes in T cells using Xdrop

Starting from just 10 ng of DNA, Xdrop reveals gene cassettes inserted using lentivirus and other transduction systems.

The accuracy of gene editing and the potential occurrence of unintended rearrangements must be assessed to understand any risks, such as cassette insertion in the vicinity of an oncogene or a tumor suppressor. However, conventional PCR screening for editing outcomes can
overlook such unintended insertions.

In this application note, we demonstrate how Xdrop was applied to identify ~1000 CAR cassette insertion sites in a lentiviral CAR-T cell sample using long read sequencing to yield high quality candidate integration sites, which was validated by Sanger sequencing of PCR amplified CAR cassette border regions.

Reliable enrichment for short-read and long-read sequencing

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.

ε3/ε3 (ONT) ε2/ε2 (ONT) ε3/ε3 (ILL) ε2/ε2 (ILL)
Enrichment of 100 kb region (fold) 197 213 154 276
Enrichment of 10 kb region (fold) 814 1114 506 795
APOE gene mean coverage (fold) 37 50 93 117
Read average length 4491 5043 151 151
Percent reads mapped to reference 95 97 97 94

Reveal unintended edits not detected by other methods

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.

Fig insertion2

Other genomic edit methods also covered: identify the insertion site of transgenes

DNA microinjection into single-cell embryos has been used to create transgenic animal models for nearly 30 years. Suprisingly, because injected transgenes insert randomly, the exact insertion site and pattern is unknown for numerous animal lines.

Here as well, Xdrop has proven helpful. Targeted enrichment with Xdrop followed by long-read sequencing identified the unexpected insertion of a Pax8-CreERT2 sequence into chromosome 1 of a transgenic mouse line developed by the U.S. National Cancer Institute. The data showed that the  modification event resulted in a 96.5 kb deletion and a triple insertion of the construct in series with variable truncations and insertions at the borders (see figure).

Transgene Instertion Schematic(1)