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DOE OSTI · 1855311

Scalable and automated CRISPR-based strain engineering using droplet microfluidics

Iwai, Kosuke (ORCID:0000000173953521)·Wehrs, Maren·Garber, Megan·Sustarich, Jess·Washburn, Lauren·Costello, Zachary·Kim, Peter W. (ORCID:0000000324650414)·Ando, David·Gaillard, William R.·Hillson, Nathan J.·Adams, Paul D.·Mukhopadhyay, Aindrila (ORCID:0000000265137425)·Garcia Martin, Hector (ORCID:0000000245569685)·Singh, Anup K.
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Abstract

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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BibTeXRIS

Iwai, Kosuke (ORCID:0000000173953521), Wehrs, Maren, Garber, Megan, Sustarich, Jess, Washburn, Lauren, Costello, Zachary, Kim, Peter W. (ORCID:0000000324650414), Ando, David, Gaillard, William R., Hillson, Nathan J., Adams, Paul D., Mukhopadhyay, Aindrila (ORCID:0000000265137425), Garcia Martin, Hector (ORCID:0000000245569685), Singh, Anup K.. 2022-03-15. Scalable and automated CRISPR-based strain engineering using droplet microfluidics. https://doi.org/10.1038/s41378-022-00357-3

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