Monitoring the promoter activity of long noncoding RNAs and stem cell differentiation through knock-in of sgRNA flanked by tRNA in an intron

Mammalian genomes encode thousands of long noncoding RNAs (lncRNAs) that are often expressed in a tissue and cell specific manner. Therefore, a reporter that can faithfully reflect the expression or activity of lncRNAs can provide tools useful not only for uncovering the regulators of lncRNAs, but also for tracking cell fate and disease status. Here, we design a sgRNA precursor in an intron (GRIT) strategy that can monitor the promoter activity of lncRNAs. We used this strategy to report the expression of Lncenc1 and Neat1 in mouse embryonic stem cells (ESCs). Furthermore, we show that GRIT may be used to track differentiation status of stem cells. We anticipate that GRIT will be applicable in dissecting regulatory mechanisms underlying the transcription of lncRNAs, tracking cell fate switch during differentiation or disease progression and integrating the promoter activity of various RNAs for synthetic biology applications.