Homo sapiens Targeted Locus (Loci). synthetic construct

The goal of the study is to obtain in depth profiles of the dependence of expression levels induced by representative TALE- and Cas9-activators on a wide set of variations of their target binding sites in a single high-throughput sequencing experiment. An expression construct is built that contains a biased, degenerate binding site for the TALE- or Cas9-activator and also a random sequence tag embedded in the activated gene. Cells transfected with the construct library will each have a unique construct with a unique transcript tag, and when the construct is induced by a TALE or Cas9-activator, the expression level of the construct in each cell can be measured by counting instances of each cell's transcript tag in RNAseq data. The binding site from which expression was induced is not, however, in the transcript tag. Therefore, an initial sequencing of the DNA construct library is performed to allow transcript tags to be mapped back to the binding sites that induced the transcripts containing them. In this way, the levels of induction achieved by tens of thousands of sequence variants of the target binding site can be measured at once from a single RNAseq experiment of a population of cells,