What is ChemoGenix?
ChemoGenix is a genome-wide pooled CRISPR/Cas9 KO screening platform, specialized in screens in
NALM6 cells in the presence of bioactive compounds
Where is ChemoGenix located?
We are part of the Institute of Research in Immunology/Cancerology
(IRIC), Université de Montréal
(UdeM) in Montreal, Quebec, Canada
What is chemogenomics?
Chemogenomics is a powerful approach to study the response of cells to chemical perturbations.
In the context of genome-wide CRISPR/Cas9 KO screens, chemogenomics allows to identify all genes
whose knockout either sensitize or suppress the growth inhibition induced by a compound.
Why is the chemogenomic signature important?
The genetic signature obtained from a chemogenomic experiment can be used to decipher or confirm
the mechanism of action (MOA) of the compound, finding potential secondary off-target effects,
identifying potential chemo-resistance or chemo-sensitivity and finding novel gene functions
involved in the cellular mechanism targeted by the compound.
Who can take advantage of ChemoGenix services?
Currently, we offer our services only to academia or other educational and non-profit institutions. Soon we
will be able to offer our services to any entity. If you are a company, contact us to stay in
touch and get the latest news.
Why should I test my compound with ChemoGenix?
First of all, ChemoGenix offers genome-wide chemogenomic CRISPR screens at the most competitive price
you can find on the market. Furthermore you will have the opportunity to compare your results with
the ones stored in the Chemogenix database. This provides you with the unique opportunity to
understand more deeply the signature of your compound, assign MOA to uncharacterized compounds or uncover
novel gene functions
How long does it take to have my compound screened?
By the time you will send us the compound it will take us between 3 to 6 months to deliver the results.
The screen itself takes about 3 months and includes the assessment of the optimal dosage for NALM6 cells
growth inhibition (3-4 weeks), compound treatment (2 weeks), sgRNA sequences amplification (3 weekls),
sequencing (2-3 weeks) and bioinformatics analysis (1 week). Because we perform rounds of screens
(about 3/4 rounds per year), with each round lasting about 4 months, the delivery of the results will
depend whether you submitted your compound(s) prior or after the start of the round.
What are NALM6 cells?
The NALM6 cell line is derived from human pre-B lymphocytic cells belonging to a 19-year-old male patient
with acute lymphoblastic leukemia.
Why do you use NALM6 cells?
We believe that NALM6 cells display features that favor mass-screening. NALM6 cells are grown in suspension,
are small, reach high concentrations, have high knock out efficiency and can be easily infected by lentiviruses.
Why don’t you offer the screening of other cell lines?
We believe that most compounds interfering with cell growth will interact fundamentally with the same
pathways in all cell types. Also, the time and the effort required to perform a dedicated custom screen
will cause a dramatic increase in the costs and will negatively affect our throughput. Nonetheless,
we can support your lab to screen in any cell line. In this format you can still take advantage of
our expertise and we do not saturate our screening capabilities, keeping costs accessible to everyone.
For how long are NALM6 cells grown in the presence of compounds?
Cells are exposed to compounds for 8 days. In theory, the longer the exposure, the higher the sgRNA
frequencies change generating more robust scores. However, longer exposure increases the risk of
random library pool drift that introduces noise in the gene scores. We obtain consistent and
reliable results with 8 days of screening time, corresponding more or less to 7.5 NALM6 cell population
doublings. If you desire to run the screens for longer times, because your compound induces
slow-acting epigenetic changes, or if you want multiple time points, please let us know.
Can you obtain signals for genes that are essential for cell survival (essential gene)?
Yes we can. Our algorithm compares changes of sgRNAs frequencies to sets of sgRNAs of matching “levels”
of essentiality and therefore allows chemogenomic scoring of all genes, whatever the number of population
doublings reached. In short, we can detect “levels” of essentiality to provide accurate gene
enrichment/depletion scores for all genes.
Can I screen a compound if I don’t know its effect on NALM6 cells growth??
Yes you can. Prior to screening, we systematically generate dose-response growth curves to test the
ability of compounds to inhibit NALM6 cell growth. This service is included in our NALM6 chemogenomic
screening service and is necessary to establish the appropriate dose for the screen. With the dose-response
results in hand you can decide whether or not to proceed with the screen. In our experience, compounds
with minimal to no effect on NALM6 growth seldomly provide positive results although in some
sporadic cases we identified a number of synthetic lethal genes. If you choose to not proceed with
the experiments, you will be charged only for the NALM6 dose-response growth curve ($100 CAN).
How much compound should I ship to ChemoGenix?
This largely depends on its ability to cause growth inhibition in NALM6 cells. If you derive the potency
of the compound from the literature, keep in mind that a dosage obtained from growth inhibition assays
in human cell lines is often comparable to what we will obtain with NALM6 cells. Conversely, dosages
obtained from in vitro assays may overestimate the effective potency. We will screen your compound in
NALM6 cells once diluted in 70 mL of 10% FBS RPMI media. Over the course of the 8-day screen, we will
dilute cells to maintain
exponential dose and supply with more compounds so we may need to sustain the dose once diluted in a
final volume of 220 mL media. As a rule of thumb, usually 5-10 mg of compound is enough. If we
do not receive enough material, this may affect the precision of our dose-response curve pre-test and
ultimately delay the start date of your screen to the following round.
How should I ship my compound?
We prefer to receive the compound in solid form stored in microcentrifuge tubes at room temperature
(unless the compound is highly unstable). You have to indicate the exact amount of compound (in mg)
because, upon reception, we will dissolve it directly in the provided tubes to the appropriate concentration.
Why do you prefer to have compounds sent to you in solid form?
Because we can ensure that every compound has been dissolved using the same stock of solvent.
Furthermore, packages shipped in dry ice can easily be delayed at the border for long times,
possibly resulting in compound degradation.
What solvent do you use?
We routinely dissolve the compound in DMSO. Alternatively, if needed, we use water (or other
water-based solvent). It is your responsibility to inform us on the recommended solvent and the
solubility of your compound.
How many screens has ChemoGenix made to date?
We have conducted over 1,200 chemogenomic screens in NALM6 cells utilizing more than 800 distinct
compounds. A considerable number of these screenings were conducted by the same team before the official
founding of Chemogenix, as part of an academic project spearheaded by Professor Mike Tyers. He is set to publish
a forthcoming paper that will elaborate on around 400 of these screenings, which we will soon make available to the public.
Why do you systematically assess the potency of compounds in NALM6
cells before launching a screen?
We routinely assess the potency of each compound prior screening to ensure that the compound
is active in NALM6 cells and to choose the most appropriate dose for the experiment. Cell growth
is the only parameter affecting the relative frequencies of sgRNAs within our pooled sgRNA
library. Only gene knockouts favoring cell growth will be enriched with a high dose screen.
On the contrary, only the gene knockouts detrimental to cell growth will be depleted with
a low dose screen. By using an intermediate dose (close to the IC50), we can capture both
cohorts of genes in a single screen. In our experience, both sides of the genetic spectrum
are equally important to assess novel gene functions and characterize compounds. If you are
interested only in rescues (also called positive selection or suppressors), we can perform
a high dose screen sacrificing the identification of synthetic lethals. Conversely, if you
are interested only in synthetic lethals (also called negative selection or sensitizers),
we can perform a low dose screen sacrificing the identification of rescues. If you are
interested in generating many more hits on “both sides” of the genetics, you can screen
at both low and high doses.
Do you do screens based on parameters other than growth inhibition like FACS?
We have performed specialized screens in different stress conditions (such as different
temperatures or oxygen concentrations…) or based on physical cell sorting like cell attachment
or cell volume separation. We have never done FACS-based screens but the IRIC has a dedicated
FACS facility with top-of-the-line sorters and we are open to considering running this type
of screens. Alternatively, and this is the option we prefer, you can acquire any library
(AddGene provides many options), do the infection, do the tissue culture work and do the
cell sorting as you like and then let us do NGS with resulting cell pellets and bioinformatics
analysis to get gene scores as per our custom screening services.
Do you do replicates while screening?
We do not run replicates, we do one screen per condition but we do run internal controls to ensure
that the round has been run correctly. We can run replicates but this will affect the final bill as
the cost of each replicate is the same as a single experiment. We suggest initially to screen with
no replicates and to consider running replicates to increase the robustness of your signature only
if the initial experiment meets your expectations.
Will my compound provide a genetic signature in NALM6 cells?
This is a complex question for which there is no direct answer. Most compounds affecting cell
growth will have the same MOA in any cell line. In this context, even if you designed your compound
to be tissue/cell line specific, it is still possible for that compound to be active in NALM6 cells
and provide a similar genetic signature to your original model. However NALM6 cells might not be
an appropriate genetic background for all compounds. For instance the tyrosine kinase inhibitor
Imatinib/Glivec specifically targets the Bcr-Abl fusion which is absent in NALM6 cells.
Do you provide support for the data interpretation?
You will be definitively supported by our staff, who can leverage the experience acquired running
and interpreting more than 1,000 screens.
Additional bioinformatics analysis can be provided (eg: GSEA, GO enrichment, network analysis)
but this has to be discussed on a case-by-case basis. We are currently developing an online tool
to allow the comparison of signatures from different compounds.
Will my data remain private?
We offer chemogenomic screens at operational costs with the expectation to release the data
publicly after 3 years from the delivery of the results. However, we fully understand that you might still need data privacy
after a 3 year time. If ever you would prefer to keep the data private we just ask
you to sponsor and pay for another screen with a compound or gene of our choice. The results
of the sponsored screen will be immediately publicly released. Our goal is to grow the
public repertoire of chemogenomic signatures in NALM6 cells in order to support the biomedical
community.
Can I publicly release my data before the 3 years mark?
Yes you can. Just let us know and the public release of your data will be anticipated.
Do I have to tell you if I myself publish the results of the screen in a manuscript?
It is not mandatory but it would be very much appreciated. It supports the growth of Chemogenix
and facilitates the dissemination of your data.
Do I have to cite ChemoGenix in my publication?
Yes. Please cite Chemogenix with its URL address in the material & methods section.
If you think that the ChemoGenix staff also contributed intellectually to the design of some
experiments and/or to the interpretation of your data, co-authorships would be greatly appreciated.
If you need help for the materials & methods section we will be happy to help.
Can you inform me if a compound has already been screened?
Data confidentiality constraints do not allow us not to disclose what has already been screened.
At the same time we will not inform other clients that your compound has already been tested.
What are the advantages of cross-screen comparisons?
Each chemogenomic signature provides a great deal of information on the nature of the compound.
However the systematic comparison of a signature to a vast repertoire of chemogenomic data allows
for a more precise classification of the compound and facilitates the extrapolation of features
hard to infer from a single experiment.
What is the EKO library?
EKO stands for Extended Knockout Library. It is a pooled sgRNAs library composed of 278,754
different sgRNAs targeting all of RefSeq (at close to 10 sgRNAs per gene)
(PMID:29038160).