Scalable and Automated CRISPR-Based Strain Engineering Using Droplet Microfluidics

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 system’s 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 . ### Competing Interest Statement N.J.H declares financial interests in TeselaGen Biotechnologies and Ansa Biotechnologies.