How are Hap1 knockout cell lines generated?
HAP1 knockouts are engineered using the CRISPR-Cas9 system to introduce frameshift mutations into the coding sequence of genes of interest.
What type of cells are HAP1 cells?
HAP1 is a near-haploid human cell line that was derived from the male chronic myelogenous leukemia (CML) cell line KBM-7 (see Carette et al. Nature. 2011.). HAP1 cells are adherent with fibroblast-like morphology.
View our overview for more information on HAP1 cells
How are HAP1 knockouts validated?
All HAP1 knockout cell lines are validated by PCR amplification and Sanger Sequencing to confirm the mutation at the genomic level. To enable you to verify our results, we provide our sequencing result, including primer sequences.
Further to this HAP1 wild-type cells have been extensively characterized by whole genome sequencing and transcriptome analysis.
How do you culture HAP1 cells?
HAP1 cells are grown in Iscove’s Modified Dulbecco’s Medium (IMDM) in the presence of 10% fetal calf serum and penicillin/ streptomycin. Cells are passaged every 48h at a dilution of 1:10 or 1:20, depending on the initial density. More detailed information is available here.
Additionally, this information is listed on the Certificate of Analysis for the cell line.
What will I receive when I order a HAP1 knockout cell line?
Customers obtain a clonal HAP1 cell line, bearing a frameshift mutation in an exon of the gene of interest. Typically, the mutation is placed closed to the 5’end of the gene, ie. in exon 1 or 2.
The mutation is described in the clone datasheet enclosed upon shipment and can be verified by a PCR on genomic DNA followed by Sanger sequencing.
All mutant cell lines are provided as an isogenic pair with the wild type line. This provides a control for your downstream experiments
How do you know that HAP1 knockout cells don’t contain off-target modifications?
While we cannot exclude entirely the possibility of off target modifications in addition to on target cleavage, we believe it represents a relatively low and controllable risk, as a number of recent publications have demonstrated that the CRISPR-Cas9 system is to be highly specific (e.g. Cencic et al).
Further to this a collaborator group have published a paper where they examine off-targets in the HAP1 cell line and observe very low frequencies.
At Horizon, to mitigate the risk still further our in-house selection algorithms warrant that only those guide RNAs with minimal predicted off-target sites in the human genome are chosen. Further to this, scientists can control their experiments through the use of multiple, independent clones or rescue of the knockout with a wild-type cDNA.
I have never worked with HAP1 cells before. How do I know if they look correct?
Our Technical Support team is happy to provide a visual inspection of your culture if you send an image to email@example.com.
In addition, further information and example images can be found in our HAP1 Cell Culture Guidelines.
Why are there multiple HAP1 cell lines listed for my gene of interest?
When we perform the CRISPR gene editing, we often isolate multiple clones with successful frameshifts. The differences between them is often the number of bases that are inserted or deleted.
If you click on the product pages for each of the clones and scroll down the page, there is a pdf link with the datasheets attached. This describes the location of the frameshift and you can then determine which is more suitable for your needs.
Are HAP1 cell lines genetically stable (i.e. do they stay haploid)?
We have observed that haploid cell lines will spontaneously diploidise over time. If a gene has been knocked out, diploidisation will not affect knockout status, but to minimise the potential impact of diploidisation one of the following approaches is recommended:
- Work with cells at very low passage. Prepare stocks of low passage cells and revive frequently.
- Culture knockout and wild type cells in parallel and allow genomes to stabilise. Then use these high passage cells for experimental analyses.
How do I know if HAP1 cells are suitable for my studying a particular biological pathway?
Please contact us to discuss your experimental needs – internal research projects with this cell line are ongoing and we may have data we can share.
If you would like to learn more about our products then please browse our dedicated HAP1 FAQ here