RNA recording in single bacterial cells using reprogrammed tracrRNAs
. This method records the transcriptome through the random integration of RNA fragments as spacers in a CRISPR array, allowing quantitative recording of RNAs encoded throughout the genome and inference of the order of multiple stimuli based on spacer order. However, the combination of low acquisition frequencies and a large transcriptome means that massive populations of cells must be sequenced to infer the recorded RNAs, masking any single-cell phenomena.
By connecting past transcriptional events to current observations, TIGER has the potential to help answer outstanding scientific questions and enable new technological capabilities.
In the process of characterizing and using TIGER, we identified key design considerations and areas for further improvement. Successful design required a compatible Rptr target with an upstream sequence harboring C in the editing window. The selected Rptr target should not contain a PAM to prevent editing of the transcript’s genomic site, although use of a Cas9 nuclease with a strict PAM greatly reduces the likelihood of this scenario.
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