Cellular Engineering
Robust Structural Variation Detection
Chromosomal rearrangements are a source of structural variation within the genome that are prominent in human diseases, where there is extreme importance in detecting translocations and deletions. Prior to the IND filing careful analysis of your Gene and Cell therapy research approaches are vital to an IND filing. Traditional approaches have been used for detecting these inversions but are simply not robust enough to detect. Our directional Genome Hybridization™ is a single stranded hybridization method, able to detect inversions routinely, providing high resolution using our chromosome paints.
As an orthogonal analysis to PCR/sequencing techniques, dGH in-Site™ assays enable, through direct visualization, definitive measurement of the presence or absence of inserts. This includes genome-wide distribution and orientation of transgenes or inserted segments. When used in combination with our targeted probes and chromosome paints, dGH in-Site™ assays can provide quantitative, single cell on- vs. off-target outcomes of the delivery and editing process, and can be used to track the stability of the edited genome in both clonal isolates and non-clonal populations.
What is dGH in-Site™?
Single Cell, Genome-Wide Perspective
KromaTiD’s dGH in-Site™ provides whole genome tracking by inserting DNA cassettes as small as 2kb. Unidirectional dGH probes provide a single cell, genome-wide perspective of cellular engineering outcomes, including tracking of viral and non-viral mediated insert integration (CRISPR/Cas and alternative systems).
- On- and off-target integration metrics
- Detects structural variations in off target CRISPR therapies
- Clean integration data, even from complex or heterogeneous cell populations
- A unique, whole genome, orthogonal method of direct visualization of inserts, without bioinformatic prediction of outcomes
- Multi-channel fluorescence for flexible and multiplex assay design
- Available for human, murine, canine, non-human primate and CHO cells.
Offering the lowest limit of detection of integrated or genomic DNA targets by fluorescence, dGH in-Site™ is the most comprehensive tool available for researchers interested in tracking engineering outcomes in a de novo fashion.
Learn more about how dGH in-Site can transform your research >>
Figure 2: Same dGH in-Site™ assay in an edited iPSC, demonstrating both on-target and random integration of insert sequence (yellow) throughout the genome.
Channel 1: Fluorescence channels overlaid, insert and bracketing probes visible on one copy of chromosome 22 and off target inserts in multiple chromosomes
Channel 2: Yellow fluorescence channel, insert probe signals on one copy of chromosome 22 (circled) and in multiple off-target chromosomes
Channel 3: Green fluorescence channel, bracketing probe signals on both copies of chromosome 22
What is dGH SCREEN?
Single-Cell Rearrangement Event Evaluation and Numbering
dGH SCREEN™ is a single cell assay designed to monitor structural variants throughout the genome in an entirely de novo fashion. By utilizing directional Genomic Hybridization technology combined with fluorescence labeling patterns and chromosomal aggregation strategies, dGH SCREEN™ provides the most comprehensive and high-resolution karyographic analysis available.
Application Benefits of SCREEN™:
- Exchange Events including reciprocal, balanced and allelic translocations;
- Orientation Events including inversions, recombinations and sister chromatid exchanges;
- Chromosomal Gain & Loss Events including sister chromatid fusions, dicentrics/acentrics, fragmentation/chromothrypsis, polypoidy, aneuploidy, monosomy, polysomy;
- Monitoring Cellular Engineering Outcomes: Genome-wide, cell-by-cell and chromosome-by-chromosome assessment of structure, pre- and post modificatio;n
- Orthogonal Data for Sequencing: Genome-wide, confirmatory data regarding rearrangements predicted with long read and other NGS analyses;
- Structural Integrity QC: A straightforward yardstick by which to measure the relative stability of cell lines, or to screen and compare candidate cell lines, based on total genomic structural variation metrics;
- Variant Discovery: Discover previously unknown mutations by de novo assessment of single cells from patient sub-types;
- Genomic Stability Assessment: Track persistence of variants over time, passages, process variable changes, etc.
With dGH SCREEN™, researchers are able to track and localize a wide variety of chromosomal rearrangements within a 10kb limit of detection. In addition to being an unbiased tool for the detection of structural variants throughout the genome SCREEN™ offers High Resolution Analysis of DNA repair activity by monitoring hallmarks of genomic instability, including sister chromatid changes. This analysis produces vast amounts of rearrangement data which can be used to create stability and structural variance profiles for complex and heterogeneous cell populations, making dGH SCREEN one of the most comprehensive and robust karyographic assays available.
Learn more about how dGH SCREEN™ can transform your research >>
Figure 1: Example of the first step in the dGH SCREEN analysis process. A metaphase spread is hybridized with 5 color dGH probes designed to measure known and unknown variants in order to provide a whole genome survey of structural rearrangements in a single cell.
Figure 2: Step 2 in dGH SCREEN analysis. Chromosomes are sorted by color and morphology to enable localization of rearrangement events.
Figure 3: Step 3 in dGH SCREEN analysis. Chromosomes are organized karyographically and aggregate sample rearrangement data is gathered for analysis of genomic stability and structural integrity.</p