Simulating a for-loop in the human genome: Design and evaluation of recombinase genetic programs that count to three

Pluripotent cells specialize into numerous cell types by receiving external signals, making fate decisions, and executing differentiation functions - a paradigm similar to computer algorithms. While advances in biosensor design have enabled cells to respond to diverse stimuli, the ability to maintain a synthetic memory of the cell's experiences that then informs its behaviors remains elusive. Here, we developed a system for cellular memory-driven behaviors by simulating a "for-loop" that counts to three using recombinase STepwise gene Expression Programs (STEPs). STEPs were genomically integrated into human cells, genotyped through targeted nanopore sequencing, and evaluated for function through changes in fluorescent reporter expression. While all STEPs were capable of heritable memory and sequential gene expression, the STEP design using tyrosine recombinases for successive excisions (TRex) significantly outperformed the others tested. We then used live cell imaging to track TRex cells as they incremented from count zero to three and observed the successive emergence of four cell states from an initially homogeneous population. The STEPs framework provides biological memory and conditional expression capabilities that, coupled with input mechanisms such as biosensors, can start to approach a programming language for biology.