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CRISPR-engineered physical unclonable functions establish a foundational security technology for provenance attestation protocols. 

Abstract A Physical Unclonable Function (PUF) is a security primitive that exploits inherent variations in manufacturing protocols to generate unique, random‐like identifiers. These identifiers are used for authentication and encryption purposes in hardware security applications in the semiconductor industry. Inspired by the success of silicon PUFs, herein it is leverage Terminal deoxynucleotidyl Transferase (TdT), a template‐independent polymerase belonging to the X‐family of DNA polymerases, to augment the intrinsic entropy generated during DNA lesion repair and rapidly produce genetic PUFs that satisfy the following properties: robustness (i.e., they repeatedly produce the same output), uniqueness (i.e., they do not coincide with any other identically produced PUF), and unclonability (i.e., they are virtually impossible to replicate). Furthermore, a post‐sequencing feature selection methodology based on logistic regression to facilitate PUF classification is developed. This experimental and computational pipeline drastically reduces production time and cost compared to conventional genetic barcoding without compromising the stringent PUF criteria of uniqueness and unclonability. This results provide novel insights into the function of TdT and represent a major step toward utilization of PUFs as a biosecurity primitive for cell line authentication and provenance attestation.