Identify CAR inserts, validate CRISPR edits, and more

Xdrop and Xdrop Sort help to reveal intended and unintended on-target and off-target rearrangements with applications in mapping CAR cassette insertion, validating CRISPR edits, and more.

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 applications of Xdrop technology

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.

Simplifying transgene insertion analysis with Xdrop® Sort

Additive transgenesis by microinjection of the gene of interest into the pronucleus of fertilized eggs is an affordable and reliable way to generate transgenic animals. Unfortunately, identifying the transgene insertion site and pattern remains challenging with existing technologies.

The Xdrop Sort workflow simplifies the process of identifying transgenes and characterizing transgene insertion sites. Just a single primer set is needed to enrich long DNA fragments containing the transgene and the neighboring chromosomal region. Encapsulation and the sorting of biological material are done on Xdrop Sort without the need of additional instruments.

In this application note, we used Xdrop Sort to enrich and Oxford Nanopore Technologies to sequence DNA from the insertion(s) and the flanking genomic region(s).  This allowed the characterization of both the insertion regions and the insertion patterns.

Reliable DNA 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 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.

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

Current CRISPR–Cas9 editing checkpoints focus on relatively small unintended edits in the immediate vicinity of intended modifications. However, large deletions and complex rearrangements can occur. Conventional PCR screening strategies can easily overlook such changes, particularly if they affect only one of the alleles.

Here, we examined cell lines modified by CRISPR-Cas9 and uncovered an unintended insertion of a co-transfection plasmid immediately downstream from the edit sites. The resulting 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

For more infomation, contact us

Please enter a valid value

Please enter a valid value

Please enter a valid value

Please enter a valid value

Please enter a name

Please enter last name

Please enter valid email

Please enter a company

Please enter country

Please enter a valid value

Please check this box