In Vitro Validation of DNA-Diffusion and Control Sequences within the AXIN2 Locus [mRNA]

DNA-Diffusion is a novel generative approach leveraging diffusion probabilistic models for the design of cell type-specific DNA regulatory sequences. To evaluate the capacity of DNA-Diffusion sequences to endogenously alter AXIN2 transcription, we employed a novel MPRA-like system that utilizes CRE-recombinase mediated cassette exchange and long-read sequencing to measure the gene's transcriptional output in response to enhancer sequences several kilobases away. We evaluated a total of 100 sequences using the AXIN2 endogenous replacement experiment using the following sequence groups: GM12878 DNA-Diffusion, GM12878 Positive Controls, GM12878 Negative Controls, K562 DNA-Diffusion, HepG2 DNA-Diffusion, and shuffled GM12878 DNA-Diffusion. Our findings demonstrate the potential of DNA-Diffusion to design sequences with therapeutic potential, showing their effectiveness in an endogenous setting. Overall design: To validate enhancer sequences in the AXIN2 locus, a cell line (MEC1) was created with a stably integrated CRE recombinase and an RMCE landing pad within the AXIN2 gene and perturbed the RNA expression using a library that includes various enhancer DNAs, barcodes, and LoxP/Lox2272 sites. The mRNA expressions were analyzed from barcode sequences obtained from mRNA and gDNA using NextSeq sequencing.