ecTag, a live-cell extrachromosomal DNA (ecDNA) tracking method

Background

Extrachromosomal DNA (ecDNA) is a circular DNA frequently found outside chromosomes in many cancer types. EcDNA is known to be oncogenic due to its characteristics, such as carrying oncogenes (cancer-causing genes) and causing very high oncogene copies. While recent studies revealed the clinical significance of ecDNA, our understanding of ecDNA's behavior and biological roles has been limited due to a lack of experimental technologies. The methods that ecDNA researchers have used, including computational analysis with next-generation sequencing to reconstruct ecDNA structure and cytogenetic assessment of ecDNA (metaphase FISH and Dapi-staining), could not provide ecDNA's dynamic behaviors. To overcome technical limitations and provide a better understanding of ecDNA, we established a method to label ecDNAs and trace their behaviors during cellular processes using live-cell imaging. Using this method, we could visualize ecDNA's segregation during cell division and ecDNA hub formation during the interphase of cancer cells.

Workflow

ecDNA structure identification

Whole-genome sequencing data from cancer cell lines or tumor samples can be processed with Amplicon Architect, a computational method to reconstruct ecDNA structures. Then you will get the information (genomic location and sequence orientation) of DNA segments located on the ecDNA. You will find at least one breakpoint region where two DNA segments are connected on your ecDNA.

Breakpoint selection and validation

[Breakpoint selection]

List up breakpoint sequences (around 500-1000 bp up/down-stream sequences from the breakpoint) that your ecDNA possesses.
Perform a visual inspection for each breakpoint region and check if you have potential PAM (protospacer adjacent motif) sequences near the breakpoint. Your PAM sequence (5'-NGG-3' for dCas9) should be around 8-12 bp upstream from your breakpoint junction site.
Shortlist breakpoints that have potential PAM sequences for further validation. The Amplicon Architect may not give you a 100% accurate breakpoint sequence. But it is essential to have a precise sequence to design sgRNA against the breakpoint! So, the breakpoint sequences need to be further validated.

[Breakpoint-PCR and Sanger Sequencing]

Design primer pairs (forward and reverse) for the shortlisted breakpoint. Primers should be designed to make a PCR product containing the breakpoint region.
Perform conventional PCR. Quickly check your PCR product to see if it is the right size by doing gel electrophoresis. Then, extract the PCR product (DNA fragment) from the gel.
Perform Sanger Sequencing.
Around 50 bp from both ends of the DNA fragment is inaccurate. Thus, ideally, make your primer pairs bind the area at least 200 bp apart from the breakpoint junction for both sides. So your breakpoint junction site is in the center of your PCR product with around 200 bp right/left arms. So, you do not need to worry about getting inaccurate sequence information due to the sequencing error.

[Target BP region selection]

Now you have accurate and precise sequence information for your shortlisted breakpoint.
Narrow down the candidates by a PAM sequence availability. You should check the PAM again.
Then, further narrow down the candidates by on-/off-target effect. We used CRISPOR.
Select the breakpoint that has higher on-target efficiency with minimum off-target effect.

[Dual FISH analysis]

You still want to validate whether your breakpoint (and ecDNA) is indeed extrachromosomal. Sometimes, the same breakpoint can be found on the chromosomes.
You can design two different DNA probes with different fluorophores to label each side of the genome from the breakpoint with different colors by performing FISH analysis with these two probes.
Now, you have the genomic region information for your two segments making your breakpoint. You can search available BAC clones (that can be generated as your DNA probes) for your genomic region of interest in Genome Browser. Make sure you select "full" under the "FISH Clones" drop-down menu in Genome Browser.
Find the appropriate BAC clone numbers (usually starting with RP11) and contact Empire Genomics to order BAC clone probes. You can select fluorophore too!

Imaging module construction

[Target region]

19-21 bp downstream sequences from your PAM are your target region.
The breakpoint junction site should be located in the center of your target region.
Your target region should not be found in Genome because the target region consists of two separate genomic sites that are apart in the regular genome. So, it is not searchable on BLAT.
The best design is to have your breakpoint junction within the seed sequence area (8-12 bp downstream from PAM).

[sgRNA cloning]

Order oligo for your target sequence and have overhangs at both 5' ends. (See more details in the Written Protocol at the end)
Select the sgRNA backbone plasmid (see options for component 3 under the Addgene Plasmids tab below)
Open the backbone plasmid with the BbsI enzyme.
Ligate the linearized backbone plasmid with the oligo (Backbone: Oligo ratio = 1:6 ~ 1:10)
Your backbone plasmid contains repeats of PUFBS (PUF-binding sites), which can cause self-recombination. So, culturing bacterial cells at low-temperature at low density is recommended.

ecTag delivery

You do not need any modification on Component 1 (dead Cas9) and Component 2 (Fluorescent protein expressing module). You can get those from Addgene. See the Addgene Plasmids tab below for catalog numbers!
Select Component 2 that is appropriate to your sgRNA design. If your sgRNA backbone contains PUFBSa, select a fluorescent module that expresses PUFa fused with a fluorescent protein.
Transfect your target cells with all three components together using Lipofectamine 3000. You may try other chemical transfection reagents.
You need to optimize the best transfection condition (component ratio, amount of each component, amount of reagents, etc.)
A 1:1:1 ratio for three components was used for patient-derived neurosphere cells in the 2021 Cancer Discovery paper. See the paper for detailed information.

Targeting efficiency test

[Intercellular specificity test]

Your ecTag is very specific to your target breakpoint that is unique to your model. In other words, your ecTag doesn't necessarily create fluorescent signals when you deliver them to other cell line models.
Have your target cell model and another cell model as a control. And deliver the same condition of the ecTag system to both models. And compare ecTag signals.

[Intracellular specificity test]

Now, you want to know if your ecTag signals in your target cells are indeed on-target signals.
Perform FISH analysis using one of the Dual-FISH probes (that you used for breakpoint validation) on your target cells transfected with ecTag. If you used Clover for ecTag, you can use a red Dual-FISH probe. If you used mRuby for ecTag, you can use a green Dual-FISH probe.
And see how many ecTag signal spots are colocalized with your FISH probe signals.
To avoid ecTag signal destruction, you should use a lower temperature and shorter incubation time for the FISH probe hybridization step. Find more information in the Written Protocol below.

Addgene Plasmids

For the imaging part of the first generation of ecTag, we incorporated a CRISPR-based genome imaging technology called Casilio (2016 Cell Research)! All backbone plasmids used in 2021 Cancer Discovery are available in Addgene! Further information for more modules is available in Cheng Lab.