Development Of aMTD-Cas9/sgRNA To Overcome The Current Limitations Of The CRISPR-Cas9 System Using TSDT Yongchan Choi, Heeseop Yoo, Jinwook Yang, Youngsil Choi and Daewoong Jo Cas9 Team, Cellivery R&D Institute, Cellivery Therapeutics, Inc., Seoul 03929, Korea. BACKGROUND AIM CRISPR-Cas9 system is a highly specific genome modification tool, Providing a novel cell-permeable Cas9 system by making aMTD- which is composed of Cas9 nuclease and sgRNA. In spite of their therapeutic potential in treating genetic diseases, however, the Cas9/sgRNA ribonucleoprotein (RNP) capable of direct cell delivery,
as a therapeutic gene-editing module that is practical to target clinical applicability of CRISPR/Cas9 system is limited by low cell/tissue delivery efficiency. human genetic diseases via genome modification. METHODS Cell-permeable (CP) Cas9 recombinant protein was developed by fusing sequence-optimized hydrophobic cell-penetrating peptide (CPP), namely advanced macromolecule transduction domain (aMTD), to deliver Cas9 directly into cells and tissues. GM06214 cells were treated with FITC-labeled His-NLS-aMTD323-Cas9 for 3 hrs. After incubation, cells were harvested and analyzed by flow
cytometry. RESULTS CONCLUSION REFERENCES Contact information Chung et al. (2020) Science Advances, 6: eaba 1193 Minyong Jung By using additional carrier such as Lim et al. (2013) Clinical Cancer Research, 19: 680-690 Lipofectamine, sgRNA showed gene-editing New Drug & Business Development activity with aMTD-Cas9. In cell experiments, Lim et al. (2013) Biomaterials, 34: 6261-6271 aMTD-Cas9 / sgRNA ribo nucleoprotein Lim et al. (2012) Molecular Therapy, 20: 1540-1549 Cellivery Therapeutics, Inc. (RNP) alone showed activity without jungmy@cellivery.com additional treatment with sgRNA. Jo et al.
(2005) Nature Medicine, 11: 892-898 Jo et al. (2001) Nature Biotechnology, 19: 929-933 +82-2-3151-8900 [J. Chromatin remodeling and epigenetics-1] Epigenetic Regulation Of Gene Structure And Function With Cell-Permeable Cas9 Yongchan Choi¹˙#, Heeseop Yoo¹, Jinwook Yang¹, Youngsil Choi¹˙*, Daewoong Jo¹˙* ¹RF/Cas9, Cellivery, Seoul 03929, Republic of Korea The clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 system is a highly specific RNA-guided genome modification tool, which is composed of Cas9 nuclease and single stranded guide RNA (sgRNA). In spite of their
great therapeutic potential in treating genetic diseases, however, the clinical applicability of CRISPR/Cas9 system is limited by low cell/tissue delivery efficiency. To address this issue, cell-permeable (CP) Cas9 recombinant protein has been developed by fusing sequence-optimized hydrophobic cell-penetrating peptide (CPP), namely advanced macromolecule transduction domain (aMTD), to deliver Cas9 directly into cells and tissues. CP-Cas9 showed high cell-permeability and gene-editing activity in T7 endonuclease assay. In addition, pre- treatment of lipofectamine-coated sgRNA followed by
CP-Cas9 showed nuclease activity whereas co-treating CP- Cas9 with guided RNA did not show any activity, suggesting that an efficient delivery of sgRNA, possibly more by aMTD conjugation, can be critical for gene-editing activity of Cas9 system. Taken together, this study provides a novel cell-permeable Cas9 system capable of direct cell delivery, as a therapeutic gene-editing module that is practical to target human genetic diseases via genome modification. RORa is crucial for attenuated inflammatory response to maintain intestinal homeostasis Dongha Kim , Se Kyu Oh and Daechan Park *,
Sungsoon Fang *,Sung Hee Baek * 3 4 2 2 1 1 Department of Anatomy, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea 2 Creative Research Initiatives Center for Epigenetic Code and Diseases, School of Biological Sciences, Seoul National University, Seoul 08826, Korea 3 Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 06273, Korea 4 Biological Sciences, Ajou University, Suwon 16499, Korea, *Corresponding author. ABSTRACT Retinoic acid receptor-related receptor alpha (RORa) functions as a transcription factor for various biological
processes including circadian rhythm, cancer, and metabolism. Here, we generate intestinal epithelial cell (IEC)-specific RORα-deficient mice (RORa ΔIEC ) and find that RORa is crucial for maintaining intestinal homeostasis by attenuating NF-kB transcriptional activity. RORa ΔIEC mice exhibit excessive intestinal inflammation and highly activated inflammatory responses in the dextran sulfate sodium (DSS) mouse colitis model. Transcriptome analysis reveals that deletion of RORa leads to upregulation of NF-kB target genes in IECs. Chromatin immunoprecipitation analysis reveals co-recruitment of
RORa and histone deacetylase 3 (HDAC3) on NF-kB target promoters and subsequent dismissal of CREB binding protein (CBP) and bromodomain-containing protein 4 (BRD4) for transcriptional repression. Together, we demonstrate that RORa/HDAC3-mediated attenuation of NF-kB signaling controls balance of inflammatory responses, and therapeutic strategies targeting this epigenetic regulation could be beneficial to the treatment of chronic inflammatory diseases including inflammatory bowel disease (IBD). 1-A 1-B 3-A 3-C 3-D 3-E 1-C 1-D 3-B 3-F Fig 1. RORa attenuates intestinal inflammation to maintain
intestinal homeostasis during DSS-induced colitis. (A) Changes in body weight of RORa and RORa ΔIEC mice treated with DSS. (B) Colon f/f length was measured in RORa f/f and RORa ΔIEC mice on day 0 (n = 3 per group) and 14 (n = 7~10 per group) after DSS treatment. (C) Representative images of H&E staining of colon sections from RORa and RORa ΔIEC mice after 0, 5 and 8 days of 2 % DSS. (D) f/f Representative bright field images of enteroids cultured for 7 days. 2-A 2-B Fig 3. RORa/HDAC3 dismiss BRD4/CBP on the NF-kB target promoters for the attenuation of intestinal inflammation. (A)
Co-immunoprecipitation assay of RORa with p65 was conducted in nuclear fractions of f/f IECs from RORa mice after 8 days of 2 % DSS. (B) Effect of overexpression of RORa on 3x kB-RE-luciferase reporter activity. (C) qRT-PCR analyses of RORa -dependent and RORa-independent NF-kB target genes (upregulated genes after 2 % DSS) in IECs from RORa and RORa ΔIEC mice after 8 days of 2 % DSS. (D) Co-immunoprecipitation assay of f/f HDAC3 with p65 and RORa was conducted in nuclear fractions of IECs from RORa and f/f RORa ΔIEC mice after 8 days of 2 % DSS. (E) Co-immunoprecipitation assay of p65 with
2-C CBP and BRD4 was conducted in nuclear fractions of IECs from RORa and RORa ΔIEC f/f mice after 8 days of 2 % DSS. (F) ChIP assays were performed on the Il-1b and Tnfa promoters in IECs from RORa and RORa ΔIEC mice after 8 days of 2 % DSS. f/f Conclusion 2-D Fig 2. Genome-wide transcriptome analysis of genes affected by RORa depletion in IECs. (A) Flow chart showing the strategy of RNA-sequencing (RNA-seq) analysis. (B) Heatmap of k-means clustering of top 5 % variably expressed genes (n= 2,668). Genes were grouped into eight clusters on the basis of expression similarity. (C) Functional
gene ontology (GO) analysis of cluster 1. (D) Transcription factor enrichment analysis in cluster 1. [J. Chromatin remodeling and epigenetics-2] RORα is crucial for attenuated inflammatory response to maintain intestinal homeostasis Dongha Kim¹,#, Se Kyu Oh²,#, Daechan Park⁴,*, Sungsoon Fang³,*, Sung Hee Baek²,* ¹Department of Anatomy, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea, ²Creative Research Initiatives Center for Epigenetic Code and Diseases, School of Biological Sciences, Seoul National University, Seoul 08826, Korea, ³Severance Biomedical Science
Institute, Yonsei University College of Medicine, Seoul 06273, Korea, ⁴Biological Sciences, Ajou University, Suwon 16499, Korea Retinoic acid-related orphan receptor α (RORα) functions as a transcription factor for various biological processes, including circadian rhythm, cancer, and metabolism. Here, we generate intestinal epithelial cell (IEC)-specific RORα- deficient (RORαΔIEC) mice and find that RORα is crucial for maintaining intestinal homeostasis by attenuating nuclear factor κB (NF-κB) transcriptional activity. RORαΔIEC mice exhibit excessive intestinal inflammation and highly
activated inflammatory responses in the dextran sulfate sodium (DSS) mouse colitis model. Transcriptome analysis reveals that deletion of RORα leads to up-regulation of NF-κB target genes in IECs. Chromatin immunoprecipitation analysis reveals corecruitment of RORα and histone deacetylase 3 (HDAC3) on NF-κB target promoters and subsequent dismissal of CREB binding protein (CBP) and bromodomaincontaining protein 4 (BRD4) for transcriptional repression. Together, we demonstrate that RORα/HDAC3-mediated attenuation of NF-κB signaling controls the balance of inflammatory responses, and therapeutic
strategies targeting this epigenetic regulation could be beneficial to the treatment of chronic inflammatory diseases, including inflammatory bowel disease (IBD). Lysine-specific demethylase 3A is important for autophagic occurrence Jisu Park, Minsol Jeon, Hyunkyung Kim Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul 02841, Republic of Korea ABSTRACT Autophagy is an essential process to maintain cell survival and homeostasis under various stress conditions. Here, we report that lysine-specific demethylase 3A (KDM3A) plays an important role in
starvation-induced autophagy. Using Kdm3a knockout mice, we demonstrate that KDM3A is crucial for proper hepatic autophagy in vivo. Hepatic mRNA expression analysis and ChIP assay in WT and Kdm3a knockout mouse livers reveal that KDM3A activates autophagy genes by reducing histone H3K9me2 levels upon fasting. Together, our finding represents previously unidentified function of KDM3A as a key regulator of autophagy, implicating potential therapeutic approaches for autophagy-related diseases. INTRODUCTION Autophagy works moderately as a basal state and can be further induced by various signals,
including nutrient starvation [1]. The acute and rapid response of autophagy mainly occurs in the cytoplasm, but recent accumulating evidence has highlighted that prolonged starvation triggers transcriptional and epigenetic regulatory programs in the autophagic process [2]. Histone methylation and demethylation are associated with these transcriptional modulations of autophagy and lysosomal genes [3.4]. KDM3A, a member of Jumonji domain-containing protein, is explicitly known to demethylate H3K9me1 and H3K9me2 and requires Fe (II) and -ketoglutarate for catalytic activity, which functions as
a transcriptional coactivator [5]. Here, we provide a functional link between transcriptional regulation of autophagy and histone demethylation. KDM3A is induced upon glucose starvation and removes H3K9me2 for transcriptional activation of autophagy genes. Further, we applied Kdm3a KO mouse model to understand how KDM3A functions as a crucial player for proper hepatic autophagy in vivo. RESULTS Figure 2. KDM3A is critical for autophagosome formation and lysosomal function (A) GFP-LC3 was transfected in WT and Kdm3a KO MEFs and the formation of GFP-LC3 punctation was examined by confocal
microscopy. GFP-LC3 (green); DAPI (blue). Graphs show quantification of LC3-positive punctate cells. Values are expressed as mean ± s.d. of three independent experiments. ***p<0.001. Scale bar, 10 μm. (B) GFP-LAMP1 was transfected in WT MEFs and Kdm3a KO MEFs, and the activation of lysosome by increased GFP-LAMP1 intensity was examined by confocal microscopy. GFP-LAMP1 (green); DAPI (blue). Graphs show quantification of intensity of LAMP1 (right). Values are expressed as mean ± s.d. of three independent experiments. ***p<0.001. Scale bar, 10 μm. (C) WT MEFs and Kdm3a KO MEFs were deprived of
glucose for 12 hours in the absence or presence of bafilomycin A1 (
