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.
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 canoverlook 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.
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.
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.
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.
Please enter a valid value
First name Please enter a name
Last name Please enter last name
Email Please enter valid email
Company Please enter a company
Country - Afghanistan Albania Algeria Argentina Armenia Australia Austria Azerbaijan Bahrain Bangladesh Belarus Belgium Belize Bhutan Bolivarian Republic of Venezuela Bolivia Bosnia and Herzegovina Botswana Brazil Brunei Darussalam Bulgaria Cambodia Cameroon Canada Chile Colombia Congo (DRC) Costa Rica Côte d’Ivoire Croatia Cuba Cyprus Czech Republic Denmark Dominican Republic Ecuador Egypt El Salvador Eritrea Estonia Ethiopia Faroe Islands Finland France Georgia Germany Greece Greenland Guatemala Haiti Honduras Hong Kong S.A.R. Hungary Iceland India Indonesia Iran Iraq Ireland Islamic Republic of Pakistan Israel Italy Jamaica Japan Jordan Kazakhstan Kenya Korea Kuwait Kyrgyzstan Lao P.D.R. Latin America Latvia Lebanon Libya Liechtenstein Lithuania Luxembourg Macao S.A.R. Macedonia (FYROM) Malaysia Maldives Mali Malta Mexico Moldova Mongolia Montenegro Morocco Myanmar Nepal Netherlands New Zealand Nicaragua Nigeria Norway Oman Panama Paraguay People's Republic of China Peru Philippines Poland Portugal Principality of Monaco Puerto Rico Qatar Réunion Romania Russia Rwanda Saudi Arabia Senegal Serbia Singapore Slovakia Slovenia Somalia South Africa Spain Sri Lanka Sweden Switzerland Syria Taiwan Tajikistan Thailand Trinidad and Tobago Tunisia Turkey Turkmenistan U.A.E. Ukraine United Kingdom United States Uruguay Uzbekistan Vietnam Yemen Zimbabwe Please enter country
Your message* Please enter a valid value
I consent to receiving communication from Samplix about products, events and industry updates. Please check this box
