Scalable and automated CRISPR-based strain engineering using droplet microfluidics

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.

Keywords: Engineering; Microfluidics.

Fig. 1. The microfluidic chip enables CRISPR-MAGE recombineering in an automated and multiplexed manner.

a CRISPR-MAGE steps. The ones inside the box are performed on the chip. Cells are removed from the chip after the recovery step for induction and plating. b The microfluidic chip in a 3D printed holder (left), the electrode pattern (right), a top-view of an individual well, and a side-view schematic of a well. The chip is designed to contain 100 discrete reaction chambers with individually addressable electrodes for multiplexed CRISPR-MAGE recombineering, and its 384-well format design can be interfaced with lab automation equipment. c Droplets containing plasmids and cells are dispensed into each chamber through the inlet port, mixed by electrowetting, and electroporated by applying a voltage pulse

Fig. 2. The microfluidic chip is capable of loading/mixing/electroporation in each chamber, enabling 100 discrete reactions on a single chip.

a Sample volumes and loading sites can be programmed as desired. Green squares show where droplets were dispensed, and the numbers show the volume dispensed in nL. b Each chamber can be loaded with droplets containing different samples, and the droplets are suspended in oil containing a surfactant to prevent evaporation and accidental merging. c Electrowetting enables on-demand sample mixing by merging droplets. d The optimal voltage required for maximum transformation is dependent on the gap between electrodes. In the final chip design, we used 100 µm gap electrodes. e One hundred parallel electroporation reactions achieved over 80% successful transformation of E. coli with GFP plasmids. GFP was expressed by induction with IPTG and confirmed by measuring the fluorescence intensity (excitation/emission = 460 nm/515–535 nm)

Fig. 3. The microfluidic chip was able to use CRMAGE to disrupt galK with an over 98 ± 3% success rate.

a Mutants from on-chip electroporation were plated on MacConkey agar plates: white colonies indicate successful galK disruption, while red colonies indicate the wild type. b The killing rate of the wild type (WT) is similar between the on-chip process and the benchtop process, with both exceeding the 90% success rate. Killing rates are calculated by counting the white colonies (i.e., mutants with galK disruption) as a proportion of the total number of colonies, including the red colonies (i.e., wild type). Error bars denote the standard deviation of biological replicates (N = 16 for the on-chip process and N = 6 for the benchtop process). c CRMAGE mutation for galK disruption was verified by Sanger sequencing. One of the mutations in the oligo produces galK disruption, while the other mutation is required for Cas9 selection since it is in the PAM region

Fig. 4. The microfluidic chip allows automated genome modification resulting in indigoidine production changes.

a Two genetic targets in the indigoidine pathway (bpsA/sfp), and four targets that affect the supply of glutamine, a precursor for indigoidine production (glnA), were selected. b Indigoidine-producing strain in the right cuvette produces a blue color with the highest absorbance at 615 nm. c The genetic modifications impact the production of indigoidine (quantified by normalizing absorbance at 615 nm by 800 nm to minimize any background noise). Error bars denote the standard deviation of biological triplicates