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

Cell Discovery Zhao and Wang Cell Discovery (2021)7:45 https://doi.org/10.1038/s41421-021-00272-3 www.nature.com/celldisc CORRESPONDENCE Open Access Monitoring the promoter activity of long noncoding RNAs and stem cell differentiation through knock-in of sgRNA flanked by tRNA in an intron 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Yu-Ting Zhao1 and Yangming Wang 2 Dear Editor, The majority of mammalian genome is transcribed to RNA transcripts, of which only a very small percentage code for proteins1. As a result, thousands of RNAs that do not code for proteins are produced in cells, including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). These noncoding RNAs exert regulatory functions in various physiological and pathological conditions2. In addition, numerous noncoding RNAs are expressed in a tissue- and cell-specific manner1. Thus, a reporter that faithfully reflects the expression or activity of noncoding RNAs can provide useful tools not only for uncovering the regulators of noncoding RNAs, but also for tracking cell fate and disease status. Previously we have designed a miRNA inducible CRISPR-Cas9 platform that can serve as a sensor for miRNA activities3. However, designing a reporter for long noncoding RNAs has not been easy due to its untranslatable nature and low expression level. Here, we design an sgRNA precursor in an intron (GRIT) strategy that can monitor the promoter activity of lncRNAs (Fig. 1a). Furthermore, we show that GRIT can be used to track differentiation status of stem cells. The design of GRIT includes three key elements (Fig. 1a): dCas9-VPR expressed under the control of a constitutively active CAGGS promoter3, an RFP gene under the control of a tetracycline-inducible promoter (TRE)3, and a transfer RNAGln (tRNAGln)4-flanked sgRNA that is integrated in an endogenous noncoding RNA locus through homologous Correspondence: Yangming Wang () 1 Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China 2 Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, China recombination. To minimize the impact of tRNA-sgRNA knock-in on lncRNAs, we chose genome region that will be expressed as an intron to knock-in tRNA-sgRNAs. In addition, for lncRNA gene without an intron, an artificially designed mini-intron-containing tRNA-sgRNA fusion sequence was knocked in. The tRNA flanking design was chosen based on our observation that tRNA-flanked sgRNA induced higher level of RFP expression when compared to ribozyme-flanked sgRNAs (Fig. 1b, c; Supplementary Fig. S1a-c and Table S1). We then knocked the tRNA-flanked sgRNA into the second intron of Lncenc1 in mouse embryonic stem cells (ESCs) in which CAGGS-dCas9-VPR and TRE-RFP have been transgenically integrated (Fig. 1d; Supplementary Table S1). Lncenc1 is a lncRNA specifically expressed in mouse ESCs5. In ESCs with GRIT successfully integrated (Lncenc1-GRIT ESCs), we observed high level of RFP expression (Fig. 1e, f). In addition, the knock-in of tRNAsgRNA have little effect on the expression of Lncenc1 and pluripotency genes including Nanog, Oct4 (also known as Pou5f1) and Sox2 (Supplementary Fig. S2a, Tables S2 and S3). Importantly, the transcription activity of Lncenc1 locus was found not affected based on qPCR analysis of precursor RNA of Lncenc1 (Supplementary Fig. S2a). Lncenc1 is downregulated during ESC differentiation5. To check whether GRIT can report the expression of Lncenc1 during ESC differentiation, we induced differentiation of Lncenc1-GRIT ESCs with all-trans retinoid acids (ATRA) and measured RFP expression during differentiation process. Interestingly, RFP was significantly decreased upon ATRA induced differentiation (Fig. 1g, h; Supplementary Fig. S2b). More importantly, RFP level was highly correlated to the RNA level of Lncenc1 (Supplementary Fig. S2c). © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Zhao and Wang Cell Discovery (2021)7:45 Fig. 1 (See legend on next page.) Page 2 of 4 Zhao and Wang Cell Discovery (2021)7:45 Page 3 of 4 (see figure on previous page) Fig. 1 GRIT monitors the promoter activity of lncRNAs and stem cell differentiation. a Schematic design of GRIT reporter system. GRIT cassette refers to pre-sgRNAs located in an intron. After the transcription of host gene, removal of flanking RNA cleavage sequences leads to the maturation of sgRNA, which activates the downstream CRISPR-on reporter system. Hammerhead, HDV and Twister ribozyme sequences are in Supplementary Table S1. b Representative microscopy images showing RFP and GFP expression in HEK293T transfected with dCas9-VPR, TRE3G-RFP, and GRIT-GFP plasmids. For control, GFP plasmid without any sgRNA cassette in the intron was transfected with dCas9-VPR and TRE3G-RFP plasmids. The schematic for the design of this experiment is shown in Supplementary Fig. S1a. Scale bars, 200 μm. The experiments were repeated three times independently with similar results. TsgT, tRNA-flanked sgRNA. WsgW, Twister ribozyme-flanked sgRNA. c Quantification of mean RFP and GFP intensity of b. Shown are mean ± SD, n = 3 independent experiments. The P-value was calculated by one-way ANOVA with two-tailed Tukey’s multiple comparisons test. d Schematic of GRIT knock-in strategy for Lncenc1. After the establishment of dCas9-VPR and TRE3G-RFP transgenic mouse ESCs, the TsgT element is knocked in the second intron of Lncenc1 locus through CRISPR-Cpf1-assisted homologous recombination. e Representative images showing RFP expression in Lncenc1-GRIT ESCs. Scale bar, 200 μm. f Mean RFP intensity of Lncenc1-GRIT ESCs and control ESCs. Shown are mean ± SD, n = 3 independent experiments. The P-value was calculated using two-tailed unpaired Student’s t-test. g Representative images showing RFP expression in undifferentiated and differentiated Lncenc1-GRIT ESCs. Scale bar, 200 μm. h Quantification of mean RFP intensity during the continuous differentiation process of Lncenc1-GRIT mESCs. Shown are mean ± SD, n = 3 independent experiments. i RT-qPCR analysis of Lncenc1 expression during the differentiation process of Lncenc1-GRIT and control ESCs. Shown are me (...truncated)