Unbiased identification of nanoparticle cell uptake mechanism via a genome-wide CRISPR/Cas9 knockout screen

A major bottleneck in nanocarrier and macromolecule development for therapeutic delivery is our limited understanding of the processes involved in their uptake into target cells. This includes their active interactions with membrane transporters that co-ordinate cellular uptake and processing. Current strategies to elucidate the mechanism of uptake, such as painstaking manipulation of individual effectors with pharmacological inhibitors or specific genetic knockdowns, are limited in scope and biased towards previously studied pathways or the intuition of the investigators. Furthermore, each of these approaches present significant off-target effects, clouding the outcomes. We set out to demonstrate that an unbiased whole-genome screening approach using pooled CRISPR/Cas9 libraries can provide a robust and rapid approach to identify novel effectors of material uptake. We develop a methodology termed fast-library of inserts (FLI)-seq for library preparation and quantitative readout of pooled screens that shows improved technical reproducibility and is easier to perform than existing methods. In this proof-of-concept study we use this approach FLI-seq to identify a solute carrier protein family member SLC18B1 as a candidate transporter for polymeric micelles. Overall design: Data includes a benchmarking experiment (containing 22 total samples, including infection replicate 1 (with 12x, 25x, 35x, 50x, 70x, 100x, and 200x cell coverage samples each with technical replicates A and B) and infection replicate 2 (with 12x, 17x, 25x, 35x, 50x, 70x, 100x, and 200x cell coverage samples), and a micelle uptake screen (with two infection replicates)