- Will we be able to download the presentation?
This presentation will be available to watch on demand on the ATCC website
- Have you observed additional mutations in any of the knock-in mutant lines once they were grown in the presence of high concentrations of the targeted therapeutic drugs?
We did not maintain the isogenic line in the presence of targeted therapeutics. There is a general approach that some researchers use, whereby drug-resistant cells are selected by growing them in the presence of the therapeutic drug of interest. In that case, the resistant model is a result of the selective killing of the sensitive subpopulation. By contrast, ATCC used the approach of creating drug resistance via knocking in the known resistance mechanism. ATCC isogenic lines were generated from a single clone after targeted CRISPR gene editing. In our studies, we used drug treatment as a means to validate the bio-functions of the isogenic models. There is very little chance that the high concentration of drug, in the short period of the challenge, will physically cause additional specific drug-resistant mutants within the single clones.
- Is it possible to use isogenic cell lines in xenograft models?
We have not carried out in vivo studies using isogenic cell lines. However, many of the parental lines that we selected for gene editing have been widely used to generate xenograft models, such as A375 (ATCC® CRL-1619™), A549 (ATCC® CCL-185™), and U-87MG (ATCC® HTB-14™). Therefore, the newly developed isogenic lines are most likely to be suitable for in vivo studies.
- How do you predict off-target sites?
To determine potential off-target effects, we generated a list of top off-target sites by using in silico computational scoring tools that predict likely cleavage sites in the genome based on sequence similarity to the on-target designed sgRNA sequence. Also, you can use the Basic Local Alignment Search Tool (BLAST) to assess the target sequences to predict off-target sites. In addition, off-target sequences will not be targeted by CRISPR/Cas9 if they do not possess a PAM (NGG) sequence. Finally, it is recommended to choose target sequences with mismatches in the 8-14 bp at the 3’ end of the target sequences.
- I am interested in modifying G-protein coupled receptors, but I would like to use your human telomerase reverse transcriptase (hTERT)-immortalized primary cells. Since these cells have a limited proliferative potential, do you have strategies to use CRISPR-guided modification on these types of cells?
We have observed variable gene editing rates even within traditional continuous cell lines. Factors such as cell proliferation potential, cell culture under single clone conditions, transfection efficiency, and the targeted-gene locus may impact the success rate. Optimization methods and strategies will vary among individual projects. For cells that have low transfection efficiency rates, we recommend using a lentiviral vector or comparable systems.
- I am interested in the 3D growth method of A549 cells that was mentioned in the webinar. Can you provide the protocol to me?
We have used InSphero GravityTRAP™ ULA Plate and Corning® Spheroid Microplates. The A549 cell line and EML4-ALK A549 isogenic line (ATCC® CCL-185IG™) were able to form spheroids in both of the 3D culture plates using the growth media recommended by ATCC. You can obtain the related 3D culture protocols from InSphero and Corning®.
- Can you preserve an isogenic cell line for long periods of time?
The isogenic lines were created by a permanent genetic modification at the targeted gene locus within the genome. Consequently, these isogenic cell lines are stable and able to maintain their bio-functions and characteristics without being grown in a drug selection culture environment. To further optimize your cells, we recommend the use of authenticated low passage, validated, high viability cells. Also, follow ATCC culturing procedures to maintain cells and conduct your assays. It is a general recommendation and good practice not to culture cells for a long period of time or over many passages. Finally, if the cells are not performing well, have been in culture too long, or are over-passaged, use a new vial of cells that has been validated and tested to be authentic from a trusted source like ATCC.
- For the B-Raf inhibitor-resistant model, are the knock-in mutations homozygous or heterozygous?
Both of the B-RAF inhibitor-resistant melanoma models, NRASQ61K and KRASG13D mutant isogenic lines (ATCC® CRL-1619IG-2™ and CRL-1619IG-1™, respectively), contain heterozygous mutations. This is consistent with what has been commonly observed in clinical tumor samples.
- What does junctional PCR mean?
Junctional or junction PCR is the PCR amplification across two adjoining segments of DNA. In this webinar presentation, junctional PCR refers to using PCR to amplify the junctional region in the targeted-gene locus. For example, although the EML4 and ALK genes are located at two different gene loci on chromosome 2 in normal status, a junction region can be formed between EML4 intron 13 and ALK intron 20. This occurs when a section of the ALK gene translocates and fuses with a section of EML4 under pathological conditions (e.g., non-small cell lung cancer). Here, we used junctional PCR to confirm the desired translocation; we performed PCR using one primer located at EML4 gene locus and the other primer located ALK gene locus. The translocation event is verified because the PCR amplicon can be generated only when there is a fusion/junction between the two genes.
- Do you use Real-time PCR (qPCR) to perform mRNA level validation?
When gene editing leads to a point mutation, we use next-generation sequencing and Sanger sequencing methods to validate mutations at the genomic DNA level and mRNA level. The target gene expression and protein expression do not change in this case. When gene editing leads to a genetic variant that can affect protein expression, such as the EML4-ALK fusion, we use qPCR and western blot to validate target gene expression and protein expression, in addition to sequencing verification.
- How do you test for off-target effects?
To determine potential off-target effects, we generated a list of top off-target sites using in silico computational scoring tools, which predict the likely cleavage sites in the genome based on sequence similarity to the on-target sgRNA sequence. There are many freely available web-based prediction tools. We use several tools to cross-reference the predicted off-target sites within the genome. We also compare the given score for the predicted off-target sites in the report. Below is a list of several computational tools:
Design sgRNA: (Broad Institute) http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design
CRISPR Design: http://crispr.mit.edu:8079/
sgRNA Scorer 2.0: https://crispr.med.harvard.edu/
- What was your experience with ss-oligos, commonly used in techniques such as CRISPR-ChAP-MS? Do you have experience in working with other types of CRISPR/Cas9 techniques?
We have used ss-oligos, ds-oligos, and plasmid donors in our projects. Overall, we saw more consistent results from plasmid donors. The ds-oligos have delivered a better outcome than ss-oligos in our hands. Although we have used Cas9 mRNA, Cas9 protein, Cas9 plasmid, and in vitro-transcribed sgRNA, we have not fully explored the other techniques such as CRISPR-ChAP-MS, CRISPRi, and CRISPRa for new cell line model development work at this point.
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