dGH in-Site gives researchers the ability to directly visualize and characterize rearrangements at their edit sites. Using synthetic oligo probes and repeat-free bioinformatic design, dGH in-Site is capable of tracking edit site translocations, inversions, copy number variants, viral integrations and other complex rearrangements with an approximate 2kb resolution.
In-site allows researchers to examine their edits one cell at a time without having to worry about PCR biases or misrepresentative sequencing averages from heterogeneous cell populations. Instead, as an orthogonal and complementary technique, in-Site helps provide the most comprehensive data available to answer the question that matters most – is my edit safe?
1/4 -Example of in-Site and SCREEN combination assay in unedited human fibroblast control line cell. Bracketing in-Site probes on chromosome 22 (circled) to identify insert target location and dGH SCREEN paints on chromosomes 1, 2, and 3 (pink). No insert would be expected in this control line.
2/4 – Example of a single cell from the same in-Site assay in an edited iPSC viewed in multiple fluorescence channels. Left image shows all fluorescence channels overlaid, middle shows yellow/insert probe channel, and right shows green/on-target bracketing probe channel. Multiple inserts are visible outside of the on-target bracketing probes, indicating high levels of off-target integrations in this cell.
3/4 – Example of the same in-Site assay in an edited iPSC line cell with no off target insertions. Target site bracketing probes in green and insert probe in yellow zoomed in and expanded on the right. Results from this sample indicated that 6% of cells had no off-target insertions.
4/4 – Image from a dGH in-Site assay in polyploid, dual-edited, HEK-293FT cells demonstrating both normal and rearranged edit site structures. At the normal edit sites (A), both sets of in-Site probes are located on the same side of the chromosome. An inversion at the edit site is also visible (B), with one portion of the in-Site probe switching orientations and showing up on the opposite chromatid from the other portions of the in-Site probe. A partial deletion at one edit site is also observed (C), with the red portion of the in-Site probes missing.
On and Off-Target Data in a Single Assay
dGH SCREEN utilizes KromaTiD’s single stranded, synthetic probes to cover every available sequence in the genome for the ultimate unbiased assessment of editing-associated rearrangements. By spanning entire chromosomes, dGH SCREEN enables researchers to directly visualize structural rearrangements anywhere in the genome, making it possible to monitor potential off-target effects without knowing where to look for them in the first place.
Used by itself or combined with dGH in-Site, SCREEN provides both on and off-target data, including translocations, inversions, copy number variants and other complex rearrangements in a single assay.
1/6 – Image of cell from an unedited control line of HEK293FT cells. In this assay, dGH paints were used on the edited chromosome 17 (yellow), and on unedited chromosomes 1, 2, and 3 (pink). No editing-associated rearrangements were observed or expected in this cell line.
2/6 – A cell from the same assay demonstrating one on target inversion (A), one translocation from the target chromosome to an off-target chromosome (B), and elevated rates of off-target inversions and translocations compared to the control line (C).
3/6 – Image of cell from an unedited control line of HEK293FT cells. In this assay, dGH paints were used on exclusively unedited chromosomes to monitor off-target effects of a CRISPR editing system. Chromosomes 1 (green), 2 (pink), and 3 (yellow) were all painted and assessed for rearrangements.
4/6 – A cell from the same assay demonstrating off-target translocations between chromosome 2 (pink) and chromosome 1 (green).
5/6 – Image of untreated control cell from dGH SCREEN assay designed to monitor CRISPR editing effects in T-cells. Chromosomes 1, 3, and 4 paints (pink) were used to track off-target rearrangements, while chromosome 2 paints (yellow) were used to analyze rearrangements on the edited chromosome. No rearrangements were observed or expected in this cell.
6/6 – Image of CRISPR treated cell from dGH SCREEN assay designed to monitor editing effects in T-cells. Chromosomes 1, 3, and 4 paints (pink) were used to track off-target rearrangements, while chromosome 2 paints (yellow) were used to analyze rearrangements on the edited chromosome. This image demonstrates an on target complex rearrangement involving a translocation and gain (yellow, circled), and an off-target inversion (pink, circled).
Ultra High-Resolution FISH Assays
By modifying hybridization conditions and eliminating daughter strand degradation, KromaTiD probes can also be used for ultra high-resolution FISH assays. In cells that rarely divide or are non-dividing, Pinpoint FISH can be used to monitor edit sites with the same resolution and applicability as dGH in-Site.
As opposed to BAC and many other FISH probes with limits of detection around 10kb, Pinpoint FISH is capable of tracking edit site regions as small as 2kb. Pinpoint FISH probes can be used in interphase or metaphase cells with a variety of color and design options available to help you get the data you need.