Engineering chemical- and light-inducible split proteins to regulate RNA and transgene expression in mammalian cells
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Citation
Abstract
The ability to regulate expression of both endogenous genes and transgenes has profoundly impacted our understanding of cell function and cell therapy development. One powerful approach to controlling regulatory proteins is by splitting the protein and fusing genetically encoded chemical and photosensory domains to each split site. These inducible systems offer temporal and spatial control over gene expression, thus revolutionizing our ability to modulate and learn from cells. There is an urgent need for more novel gene regulatory tools that enable precise and multiplex programmable control over biological processes. Existing multi-input circuits are hampered by their type of regulation and the scale of possible outputs given the number of inputs. We addressed these limitations by expanding the tools available for RNA and gene regulation with a collection of chemical-inducible Cas13 ribonucleases and red light-inducible recombinases. While Cas13 ribonucleases modulate reversible analog transcriptomic outputs, site specific recombinases provide a complementary approach of control over permanent, transgene outputs. First, we created a suite of chemical and light-inducible Cas13 ribonucleases that served as ON and OFF switches. We also multiplexed Cas13 ribonucleases together to implement Boolean logic such as an OR gate and a demultiplexer. In parallel, we developed a suite of 10 red light-inducible recombinases. We then multiplexed the top performing red light-inducible Cre and Flp recombinases with our lab’s existing, top performing blue light-inducible recombinases. These recombinases were implemented with AND gate logic and also our Boolean Logic and Arithmetic through DNA Excision (BLADE) platform that has 2n outputs, where n is the number of inputs. We additionally patterned red and blue light-inducible recombinase outputs to demonstrate spatial control over transgene expression. Together, the suites of split Cas13 ribonucleases and split recombinases expand our capability to precisely regulate multiplexed transgenes and RNA to understand the role of multiple interacting genes in endogenous signaling networks.
Description
2025