26 Jan Droplet microfluidics for deep mutational scanning
A droplet microfluidic chip is developed as a part of a platform for deep mutational scanning of human executioner caspases.
“The human caspase family comprises 12 cysteine proteases that are centrally involved in cell death and inflammation responses. The members of this family have conserved sequences and structures, highly similar enzymatic activities and substrate preferences, and overlapping physiological roles. In this paper, we present a deep mutational scan of the executioner caspases CASP3 and CASP7 to dissect differences in their structure, function, and regulation. Our approach leverages high-throughput microfluidic screening to analyze hundreds of thousands of caspase variants in tightly controlled in vitro reactions. The resulting data provides a large-scale and unbiased view of the impact of amino acid substitutions on the proteolytic activity of CASP3 and CASP7. We use this data to pinpoint key functional differences between CASP3 and CASP7, including a secondary internal cleavage site, CASP7 Q196 that is not present in CASP3. Our results will open avenues for inquiry in caspase function and regulation that could potentially inform the development of future caspase-specific therapeutics.”
“a A schematic of our microfluidic screening system. A dilute suspension of E. coli expressing caspase variants are injected into a microfluidic device and individual cells are encapsulated into microdroplets containing lysis reagents and a fluorogenic caspase substrate. The cells are lysed, the enzyme reaction is incubated on-chip, and the fluorescence of each droplet is analyzed using a laser. The fluorescent droplets are then sorted by electrocoalescence with an aqueous stream that collects the sorted plasmids for downstream analysis. b Droplets containing active caspase variants will fluoresce, whereas empty droplets and droplets containing inactive caspases will not. c Microscopy images of droplets containing active WT CASP3 and WT CASP7 display strong green fluorescence, while droplets containing the inactive CASP3 D175A and CASP7 H144A variants remain dark. d Results of a mock screen demonstrate over tenfold enrichment of active CASP3 and CASP7.” Reproduced under Creative Commons Attribution 4.0 International License from Roychowdury, H., Romero, P.A. Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation. Cell Death Discov. 8, 7 (2022). https://doi.org/10.1038/s41420-021-00799-0
Figures and the abstract are reproduced from Roychowdury, H., Romero, P.A. Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation. Cell Death Discov. 8, 7 (2022). https://doi.org/10.1038/s41420-021-00799-0 under Creative Commons Attribution 4.0 International License
Read the original article: Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation