Scalable and Automated CRISPR-Based Strain Engineering Using Droplet Microfluidics

Iwai, K.; Wehrs, M.; Garber, M.; Sustarich, J.; Washburn, L.; Costello, Z.; Kim, P. W.; Ando, D.; Gaillard, W. R.; Hillson, N. J.; Adams, P. D.; Mukhopadhyay, A.; Martin, H. G.; Singh, A. K.

doi:10.1101/2021.06.10.447396

bioRxiv · 10.1101/2021.06.10.447396

Scalable and Automated CRISPR-Based Strain Engineering Using Droplet Microfluidics

Iwai, K.·Wehrs, M.·Garber, M.·Sustarich, J.·Washburn, L.·Costello, Z.·Kim, P. W.·Ando, D.·Gaillard, W. R.·Hillson, N. J.·Adams, P. D.·Mukhopadhyay, A.·Martin, H. G.·Singh, A. K.

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Abstract

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

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Iwai, K., Wehrs, M., Garber, M., Sustarich, J., Washburn, L., Costello, Z., Kim, P. W., Ando, D., Gaillard, W. R., Hillson, N. J., Adams, P. D., Mukhopadhyay, A., Martin, H. G., Singh, A. K.. 2021-06-10. Scalable and Automated CRISPR-Based Strain Engineering Using Droplet Microfluidics. https://doi.org/10.1101/2021.06.10.447396

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