Loss of function screening offers a hugely powerful means to characterise genetic interactions, and identify novel factors involved in any number of biological processes, from cell function to drug resistance.
Of course all screen hits require subsequent validation – and exploring these hits can be labour intensive, and so the best screens are those that minimise false positives whilst enabling even weaker true positives to be distinguished, thereby minimising time wasted in the lab.
Until relatively recently almost all loss of function screens were performed using RNAi. A major problem with this system however is a propensity for false positives caused by off-target effects, a high risk of false negatives due to incomplete knockdown, and further complications associated with reproducibility (or lack of it) across independent screens.
Alternative approaches are now available to us in the form of promoter trapping screens in haploid cells, and more recently through CRISPR screening. Both ablate gene function at the genomic level and both can produce highly significant results, giving scientists much more confidence in their hit list.
While the genetic status of haploid cells arguably makes them “not normal” (a case that could be made for most cancer cell lines), what is a weakness from some points of view is a strength when it comes to genetic screening.
The first haploid human cell line to be isolated was the KBM7 cell line that Kotecki and colleagues obtained from a male chronic myeloid leukaemia patient in 1999. But despite Kotecki highlighting the potential of haploid cells to facilitate the genetic analysis of human cells, it wasn’t until 2009 in a seminal paper in Science that their use as a screening platform was showcased.
In this paper Carette et al use a retroviral gene trapping approach to disrupt gene in a massive parallel fashion. Those pools of mutant cells could then be utilized for unbiased genetic screens, first to identify 6-TG and Imatinib (Gleevec) resistance genes, and second to isolate novel host factors essential for influenza virus infectivity and ADP-ribosylating toxins (such as anthrax).
And since then similar screens in haploid cells have been used to identify (amongst other things):
And most recently the same group responsible for debuting the haploid promoter trapping approach in 2009 has used a parallel screen in both KBM7 and HAP1s to identify the core “essentialome” of these cells. This paper from Blomen et al is the first to describe which genes are essential in human cells at the single cell level.
With the arrival of CRISPR-Cas9 screening, scientists now have the means to do genetic screening in all human cells lines. The technique is no less powerful in haploid cells however, as evidenced by one of the first published CRISPR screens which uses both HL60 and KBM7 cell lines in parallel. In this screen Wang et al, using a library of 73,000 guides to identify essential genes, find that KBM7 cells not only exhibit good concordance with the HL60 results, but exhibit a much lower signal/noise ratio.
And maintaining this low signal/noise while maximising editing efficiency will be critical for more challenging experimental paradigms such as drop out screens to look for sensitizing factors, or where multiple sites are targeted at one time, either for multiplex genome editing or to excise elements such as long non-coding RNAs.
The measure of any screen’s quality is undoubtedly the number of hits that turn out to be valid. But given the speed with which extensive, unbiased genetic screens can be performed it’s not always possible to validate every hit for every screen.
The need remains therefore to triage the hit list, and select those candidates most likely to yield interesting or important scientific breakthroughs.
Horizon’s collection of over 2000 ready-made HAP1 knockout clones gives scientists the ability to rapidly validate hits and reduce the risk of pursuing false positives. In their paper, Blomen et al use haploid knockout lines to explore the reciprocity of genetic interactions. At Horizon we have the lines to validate genes that confer resistance to 6-Thioguanine (6-TG), identified using a whole genome CRISPR screen (Figure 1).
Figure 1 - Overview of CRISPR Resistance Screen Workflow. MLH1 and MSH6 were identified s hits in a 6-TG resistance screen. Analysis of HAP1 knockout cell lines for these genes found a significant increase in 6-TG IC50 in these cell lines.
As with any cancer cell line, one cannot claim that haploid cells are suitable for studying every biological pathway or experimental scenario. But through a genetic quirk, cancer has seemingly sowed the seeds of its own demise, in the form of a highly robust screening tool.
Haploid cells may not be the perfect cell line. But are they the perfect screening background? Quite possibly!