Fig. 5: Incorporation of self-cleaving RNA ribozymes allows the use of Pol II-mediated donor cell sgRNA expression in the CROSS-FIRE system. | Nature Communications

Fig. 5: Incorporation of self-cleaving RNA ribozymes allows the use of Pol II-mediated donor cell sgRNA expression in the CROSS-FIRE system.

From: A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA

Fig. 5

a Standard expression of sgRNAs under a Pol III promoter. Transcription is ended by a Pol III terminator sequence; TTTTTT. The expressed sgRNA is not subjected to any additional post-transcriptional modifications under a Pol III promoter. b Incorporation of self-cleaving Hammerhead (HH) and Hepatitis Delta Virus (HDV) RNA ribozymes sequences flanking the sgRNA sequence expressed under a Pol II promoter results in the removal of post-transcriptional Pol II modifications. c A construct for doxycycline-inducible expression of sgRNAs using a Pol II TET-On inducible system, by incorporation of self-cleaving RNA ribozymes. d Flow cytometry analysis of Stoplight+spCas9+ HEK293T reporter cells after a five day co-culture with sgRNA+ MDA-MB-231 donor cells expressing sgRNAs under a U6 Pol III promoter (a), or a EF1a Pol II promoter using self-cleaving RNA ribozymes (b). Means + SD, n = 3 independent experiments, Tukey’s multiple comparison test. e Flow cytometry analysis of Stoplight+spCas9+ HEK293T reporter cells after a five day co-culture with sgRNA+ MDA-MB-231 donor cells expressing sgRNAs under regulation of a U6 Pol III promoter (a), and a doxycycline-inducible promoter Pol II promoter (c). Means + SD, n = 3 biologically independent samples, Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001.

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