Analyze assemblon libraries for effects on gene expression, chromatin features, and nuclear architecture.
Following assemblon/payload delivery, gene expression and chromatin architecture are probed in order to identify functional regulatory variations. Data are then fed into computational models in order to infer local and global mechanisms of transcriptional regulation. Information collected is also fed back to the design and synthesis steps in order to generate more informative assemblon libraries (see below).
Frequently, the selected gene for dissection in not sufficiently expressed in the cell type into which it is initially integrated (e.g. a neuronal gene in ESCs), and cannot be efficiently integrated directly into a more relevant cell type (e.g. neurons, that cannot be readily edited). In this case, following integration into ESCs, cells are differentiated into the desired cell type prior to gene expression analysis.
Examples of combinatorial variant libraries that can be made using Assemblon strategy:
Library designs:
Our overall strategy is uniquely suited to the study transcriptional regulation because of the important roles of genomic context, the long distances of gene regulation, and the potential to elucidate combinatorial effects. Our initial focus will be on libraries containing single, pairwise, and multiwise substitution variants that replace endogenous DNaseI hypersensitive sites. Our technology, nevertheless, can also be used to evaluate many other types of variation for functional impact, including CTCF sites, natural SNVs, retrotransposon insertions, and noncoding RNAs, which can also be moved around relative to each other, or flipped.