Taking Screening Methods to the Next Level

Institute of Molecular Biotechnology of the Austrian Academy of Sciences
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CRISPR-UMI, a novel method developed at IMBA, facilitates extremely robust and sensitive screens by tracking single mutants within a population of cells

“The whole is greater than the sum of its parts” is an adage that applies to many concepts in biology. For genetic screens, however, it is the individual parts, i.e. the individual cells, that are the focus of the next generation of CRISPR-Cas9 screens. Single mutants within a population reveal new findings that could revolutionize target discovery and offer fresh insights into the biological systems of cell differentiation and cancer.

In recent years, different labs have been working on improving CRISPR screening technology by optimizing guide efficiency, on-target specifity, and Cas9 variants. But so far, no one has been able to address the concern of cellular heterogeneity, which affects the precision of screening results. The Elling Lab has now overcome this limitation. As reported in the current issue of Nature Methods, the lab developed a new screening paradigm that allows scientists to track individual mutant cells within a screen. This breakthrough enables the detection of outliers within populations that would otherwise lead to incorrect conclusions and moreover can focus on the fraction of cells displaying a phenotype presumably due to homozygous mutation of genes by CRISPR/Cas9. It also allows to quantify the phenotype independent on the technological aspect of how efficient knockout cells are generated. The innovative method relies on the addition of a high complexity barcoding system—or Unique Molecular Identifier (UMI)—that uniquely marks each mutant clone within a population. “Instead of examining a pool of cells with variable genetic status, we can now look at hundreds of independent single cells derived clones separately. This method made it possible to reliable ascertain new drug targets for cancer therapy”, said Georg Michlits, first author.

Biological events with low probability, such as reprogramming a fibroblast to a stem cell or the formation of metastasis, can be considered to be stochastic events. A conventional screen is sensitive to the size of the event (large stem cell colony or large metastasis), but fails to assess the probability (regulation) of the event itself that is reflected in number versus size. Using CRISPR-UMI, Elling’s lab screened for genes that inhibit the reprogramming of differentiated cells to pluripotency. “We were able to directly quantify the number and size of independent iPS cell colonies that appeared in the screen, revealing whether the selected genes regulate speed or likelihood of the switch to pluripotency. CRISPR-UMI therefore enabled a direct biological interpretation of screening results,” said Ulrich Elling, group leader at the Institute of Molecular Biotechnology (IMBA).

CRISPR-UMI, a novel method developed at IMBA
CRISPR-UMI, a novel method developed at IMBA

CRISPR-UMI relies on the addition of a high complexity barcoding system—or Unique Molecular Identifier (UMI)—that uniquely marks each mutant clone within a population, thus it allows tracking single mutants within a population of cells.