Generation of barcoded stable cells To generate the barcoded stable cells, ~10 million of the cells were seeded onto a 10-cm petri dish. Sixteen hours later, the cells were transiently transfected with 1 ug of the donor plasmid (barcodetruncated CMV-mKate-PGK1-hygromycin resistance gene) and 9 ug of CMV-SpCas9-U6-AAVS1/sgRNA plasmid using the jetPRIME reagent (Polyplus Transfection). Forty-eight hours later, hygromycin B (Thermo Fisher Scientific, catalog number 10687010) was added at the final concentration of 200 g/ml. The selection lasted ~2 weeks, after which the surviving clones were pooled to generate the polyclonal stable cells. The barcoded stable cells were further expanded and maintained in the complete growth medium containing hygromycin (200 ug/ml) For transient transfection, ~300,000 cells in 1 ml of complete medium were plated into each well of 12-well culture–treated plastic plates and grown for 16 to 20 hours. All transfections were then performed using 1.75 ul of jetPRIME (Polyplus Transfection) and 75 ul of jetPRIME buffer. The transfection mixture was then applied to the cells and mixed with the medium by gentle shaking.

A physical unclonable function (PUF) is a physical entity that provides a measurable output that can be used as a unique and irreproducible identifier for the artifact wherein it is embedded. Popularized by the electronics industry, silicon PUFs leverage the inherent physical variations of semiconductor manufacturing to establish intrinsic security primitives for attesting integrated circuits. Owing to the stochastic nature of these variations, photolithographically manufactured silicon PUFs are impossible to reproduce (thus unclonable). Inspired by the success of silicon PUFs, we sought to create the first generation of genetic PUFs in human cells. We demonstrate that these PUFs are robust (i.e., they repeatedly produce the same output), unique (i.e., they do not coincide with any other identically produced PUF), and unclonable (i.e., they are virtually impossible to replicate). Furthermore, we demonstrate that CRISPR-engineered PUFs (CRISPR-PUFs) can serve as a foundational principle for establishing provenance attestation protocols.