The CRISPR/Cas9 and other genome editing techniques let us make genome wide modifications of DNA sequence or gene expression almost at will. At the same time our imaging technology allows exceptionally sensitive live cell phenotyping. BUT it is by combining genomics and biophysics that we could really revolutionize the way we do biological research.

We rose to the challenge by developing our culinary skills. The DuMPLING (Dynamic u-fluidics Microscopy Phenotyping of a Library before IN situ Genotyping) technique can be used to analyze dynamic phenotypes in large genetic libraries and subsequently recover the underlying genotype - all in one microfluidic chip.

As proof of principle we have targeted different components of the LacI operon in E. Coli using Cas9 silencing. From a plasmid library we express sgRNA for the different components together with a barcode that can be used to recover the genotype. The E. coli cells transformed with the plasmid pool are phenotyped one by one in a microfluidic chip allowing only one single clone to propagate in each position. When the phenotype has been determined, the barcodes are interrogated by sequential rounds of FISH probe hybridization.

In this study we only use a small library, but with this technique in place it is possible to scale up to libraries of several thousand genotypes. Effectively this means that we will be able to measure the effect of repressing or enhancing every single gene in the E. coli genome in one experiment. Adding all the different phenotypes that are possible to record using time lapse fluorescent microscopy, the DUMPLING might finally give us the tool that we need to understands how complex regulatory networks are integrated to achieve the high level of control that characterize all biological life forms.

The complete article is available in MSB.

Michael Lawson, the master chef, talks about the new technology.