Scalable and automated CRISPR-based strain engineering using droplet microfluidics

Iwai, Kosuke; Wehrs, Maren; Garber, Megan; Sustarich, Jess; Washburn, Lauren; Costello, Zachary; Kim, Peter W.; Ando, David; Gaillard, William R.; Hillson, Nathan J.; Adams, Paul D.; Mukhopadhyay, Aindrila; Garcia Martin, Hector; Singh, Anup K.

doi:10.1038/s41378-022-00357-3

Title: Scalable and automated CRISPR-based strain engineering using droplet microfluidics

Journal Article · 15 March 2022 · Microsystems & Nanoengineering (Online)

DOI: https://doi.org/10.1038/s41378-022-00357-3 · OSTI ID:1855311

; Wehrs, Maren; Garber, Megan; Sustarich, Jess; Washburn, Lauren; Costello, Zachary;

; Ando, David; Gaillard, William R.; Hillson, Nathan J.; Adams, Paul D.;

;

; Singh, Anup K.

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 .

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Sandia National Laboratories (SNL-CA), Livermore, CA (United States)

Sponsoring Organization:: USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231; NA0003523; NA0003525

OSTI ID:: 1855311

Journal Information:: Microsystems & Nanoengineering (Online), Journal Name: Microsystems & Nanoengineering (Online) Journal Issue: 1 Vol. 8; ISSN 2055-7434

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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