Abstract

We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on a chip. The microfluidic device contains a 10 × 10 element array, and each element contains sets of electrodes for two electric field-actuated operations: electrowetting for merging droplets to mix reagents and electroporation for transformation. This device can perform up to 100 genetic modification reactions in parallel, providing a scalable platform for generating the large number of engineered strains required for the combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the system’s capabilities through the CRISPR-based engineering of two test cases: (1) disruption of the function of the enzyme galactokinase (galK) in E. coli and (2) targeted engineering of the glutamine synthetase gene (glnA) and the blue-pigment synthetase gene (bpsA) to improve indigoidine production in E. coli.

Details

Title
Scalable and automated CRISPR-based strain engineering using droplet microfluidics
Author
Iwai Kosuke 1   VIAFID ORCID Logo  ; Wehrs Maren 2 ; Garber, Megan 2 ; Sustarich Jess 1 ; Washburn, Lauren 1 ; Costello, Zachary 2 ; Kim, Peter W 1   VIAFID ORCID Logo  ; Ando, David 2 ; Gaillard, William R 1 ; Hillson, Nathan J 3 ; Adams, Paul D 4 ; Mukhopadhyay Aindrila 2   VIAFID ORCID Logo  ; Garcia, Martin Hector 5   VIAFID ORCID Logo  ; Singh, Anup K 1 

1  DOE Joint BioEnergy Institute, Technology Division, Emeryville, USA (GRID:grid.451372.6) (ISNI:0000 0004 0407 8980); Sandia National Laboratories, Biotechnology and Bioengineering Department, Livermore, USA (GRID:grid.474523.3) (ISNI:0000000403888279) 
2  DOE Joint BioEnergy Institute, Biofuels and Bioproducts Division, Emeryville, USA (GRID:grid.451372.6) (ISNI:0000 0004 0407 8980); Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, USA (GRID:grid.184769.5) (ISNI:0000 0001 2231 4551) 
3  DOE Joint BioEnergy Institute, Technology Division, Emeryville, USA (GRID:grid.451372.6) (ISNI:0000 0004 0407 8980); Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, USA (GRID:grid.184769.5) (ISNI:0000 0001 2231 4551) 
4  DOE Joint BioEnergy Institute, Technology Division, Emeryville, USA (GRID:grid.451372.6) (ISNI:0000 0004 0407 8980); Lawrence Berkeley National Laboratory, Molecular Biophysics and Integrated Bioimaging Division, Berkeley, USA (GRID:grid.184769.5) (ISNI:0000 0001 2231 4551); University of California, Department of Bioengineering, Berkeley, USA (GRID:grid.47840.3f) (ISNI:0000 0001 2181 7878) 
5  DOE Joint BioEnergy Institute, Biofuels and Bioproducts Division, Emeryville, USA (GRID:grid.451372.6) (ISNI:0000 0004 0407 8980); Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, USA (GRID:grid.184769.5) (ISNI:0000 0001 2231 4551); Basque Center for Applied Mathematics, BCAM, Bilbao, Spain (GRID:grid.462072.5) (ISNI:0000 0004 0467 2410) 
Publication year
2022
Publication date
2022
Publisher
Springer Nature B.V.
ISSN
20961030
e-ISSN
20557434
Source type
Scholarly Journal
Language of publication
English
DOI
https://doi.org/10.1038/s41378-022-00357-3
ProQuest document ID
2639131962
Copyright
© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2022. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.