Overall, these results indicate that targeting of the exon 1B promoter does not substantially affect usage of the upstream liver-specific promoter 1D, and thus, does not dramatically reduce NEMO protein expression in a human liver cell line. Open in a separate window Fig 6 Targeting of the exon 1B core promoter with CRISPR/Cas9 does not affect NEMO expression in liver cells.(A) SNU-423 cells were transduced with a LentiCRISPR2.0-Cas9 construct containing exon 1B gRNA, and transduced cells were selected with puromycin. cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined KMT6 genomic alterations at promoter B, and do not support activation of canonical NF-B signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-B signaling is intact in these NEMO-deficient Compound K cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-B signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-B signaling are discussed. Introduction Much functional gene diversity in humans is generated by the use of alternative splicing and alternative promoters [1, 2]. It is estimated that over 50% of human genes have alternative splicing and/or use alternative promoters, and alternative promoter usage has also been coupled to alternative splicing [2, 3, 4]. In many cases, alternative promoters are used for the tissue-specific or developmentally timed expression of a given gene, and abnormal alternative splicing or promoter usage has been associated with human disease, especially cancer [2, 5, 6, 7]. For some genes, alternative promoters direct the expression of an identical protein coding region in different cell types or under different conditions by virtue of the promoters being located upstream of distinct 5 non-translated exons that splice to a common set of downstream coding exons. Methods for assessing the function of tissue-specific alternative promoter usage for individual genes are limited. Compound K In this paper, we have used a CRISPR/Cas9-based targeting approach to investigate cell type-specific promoter expression of a key gene (gene (develop liver damage and sometimes cancer [17, 18]. We had three goals in this research: 1) to demonstrate that CRISPR-based targeting of an alternative promoter can be used to knock down expression of a gene in a tissue-specific manner; 2) to create a NEMO-deficient, highly transfectable human cell line for NEMO protein analysis; and 3) to establish a proof-of-principle concept for targeting the NF-B signaling pathway for disease intervention Compound K in a way that might circumvent unwanted side effects in the liver. Results CRISPR-based targeting of a core promoter sequence in Exon 1B of the gene abolishes NEMO protein expression in HEK 293T cells The human (transcript found on polysomes in human 293T embryonic kidney cells [20] (see also Fig 1A). Within exon 1B, we noted a sequence (gene, and that is within a consensus sequence that is located near the TSS of many genes [21] (Fig 1A). Based on these cumulative observations, we put forth the hypothesis that this sequence is important for efficient transcription of the gene in 293T cells. Open in a separate window Fig 1 General structure of the 5 portion of the human gene.(A) Shown are the four 5 alternative non-coding exons (1D, 1A, 1B, 1C) of the gene on chromosome X, as determined by Fusco et al. [19]. exon 1B has RNAPII, H3K4me3 and DNase hypersensitive site Compound K footprints in HEK 293 cells (https://www.encodeproject.org/experiments/ENCSR000DTU/; https://www.encodeproject.org/experiments/ENCSR000EJR/). (B) Downstream of the exon 1B transcription start site (arrow) is a sequence (red) Compound K that aligns with a consensus motif (above the red box) that is found near transcription start site of many genes [21]. As a first step in testing that hypothesis, we sought to disrupt the predicted exon 1B core promoter element by CRISPR/Cas9 targeting in 293T cells using lentiviral transduction of Cas9 and a gRNA targeting the identified site. After puromycin selection to create a pool of transduced 293T cells, we performed Western blotting for NEMO. As shown in Fig 2A, the levels of NEMO protein were clearly reduced in two independent pools of cells transduced with the lentivirus containing the targeting gRNA as compared to cells transduced with the same vector containing no gRNA. Equal levels of total.