Jakimo, N. "Genomic nucleic acid memory storage with directed endonuclease"

Abstract

Technologies for long-term recording of cellular pathway activation are constrained by the difficultly to constantly monitor transient signaling events and expression of target genes. To overcome these limitations we designed a recording tool that uses the transcriptional output of a signaling pathway as the input for an engineered genome encoded memory. The mechanism of recording leverages the programmable nature of the bacterial immune system that consists of Clustered Regularly Interspaced Short Palindromic Repeat Sequences (CRISPR), which can recognize and cleave viral DNA using an RNA-guided directed endonuclease. Cuts left by the endonuclease are repaired by an error-prone DNA damage repair mechanism, namely non-homologous end joining (NHEJ), likely to leave mutations at the cut sites. Defining the cut site with pathway-dependent transcription of guide RNA, this genomic region is sequenced to measure pathway activation by the amount of accumulated mutations. To demonstrate a system to monitor cancer metabolism, guide RNA is expressed in mammalian cell culture with a NF-kappaB promoter. To demonstrate a system that can monitor sugar intake in an environment like the gut, guide RNA is expressed in bacteria with an arabinose promoter.