scholarly journals DExCon, DExogron, LUXon: on-demand expression control of endogenous genes reveals differential dynamics of Rab11 family members

2021 ◽  
Author(s):  
Jakub Gemperle ◽  
Thomas Harrison ◽  
Chloe Flett ◽  
Antony Adamson ◽  
Patrick Caswell
Keyword(s):  
Gene Expression ◽  
Family Members ◽  
Light Illumination ◽  
Endogenous Gene ◽  
On Demand ◽  
Endogenous Expression ◽  
Expression Control ◽  
Diploid Cells ◽  
In Cells ◽  
Expression Kinetics

CRISPR technology has made generation of gene knockouts widely achievable in cells. However, once inactivated, their reactivation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation) and LUXon (light responsive DExCon), approaches which combine one-step CRISPR-Cas9 mediated targeted knock-in of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene transcription with the ability to reactivate transcription on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knockout/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon) or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, expression kinetics and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in cells.GRAPHICAL ABSTRACTIN BRIEFWe describe development of DExCon, LUXon and DExogron approaches, where a single CRIPR/Cas9-mediated gene editing event can block endogenous gene expression, with the ability to reactivate expression encoded such that even silent genes can be expressed. Expression can be controlled systematically using doxycycline, or spatiotemporally by light, allowing fluorescent tagging of endogenous proteins and quantification of expression kinetics, protein dynamics and stability for highly similar genes such as members of the Rab11 family.

Caged siRNAs with single folic acid modification of antisense RNA for photomodulation of exogenous and endogenous gene expression in cells

2018 ◽  
Vol 16 (38) ◽  
pp. 7029-7035 ◽  
Author(s):  
Lijia Yu ◽  
Nannan Jing ◽  
Zhenjun Yang ◽  
Lihe Zhang ◽  
Xinjing Tang
Keyword(s):  
Gene Expression ◽  
Folic Acid ◽  
Complex Formation ◽  
Antisense Rna ◽  
Folate Receptor ◽  
Acid Modification ◽  
Endogenous Gene ◽  
In Cells

Photoregulating gene expression using folic acid modified caged siRNA through complex formation of folic acid/folate receptor.

60 CRISPR-on, a new tool for activation of endogenous gene expression in bovine embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 155
Author(s):  
V. Savy ◽  
V. Alberio ◽  
N. Canel ◽  
L. Ratner ◽  
M. Gismondi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Regulatory Region ◽  
Bovine Embryos ◽  
Endogenous Gene ◽  
Cell Fate Decisions ◽  
Guide Rnas ◽  
Endogenous Expression ◽  
Mammalian Embryos

The CRISPR-Cas9 system enables precise genome editing in mammalian somatic cells and embryos at a very high efficiency. A modified version of Cas9 (dCas9) was engineered, resulting in a DNA binding protein capable of site-specific target recognition but unable to cut the DNA. By means of dCas9 fusion to heterologous domains, including transcriptional activators or repressors, specific modulation of gene expression has successfully been achieved invitro, making possible the modulation of the cell-differentiation state. However, CRISPR-mediated transcriptional activation (CRISPR-on) has been mainly used invitro, and to our knowledge, there are no reports regarding its use for the activation of endogenous gene expression in mammalian embryos. As a proof of principle, we evaluated the CRISPR-on system in bovine embryos for modulation of endogenous expression of SMARCA4 and TFAP2C, transcription factors implicated in trophoblast lineage commitment. We hypothesised that CRISPR-on may induce SMARCA4 or TFAP2C endogenous expression, enabling the design of strategies to induce trophectoderm proliferation of invitro-derived embryos. To this aim, we designed and synthesised 4 non-overlapping single guide RNAs to target the regulatory region of each of these target genes. Presumptive zygotes were cytoplasmically microinjected with a mix containing dCas9-VP160 mRNA and a pool of 4 single guide RNAs targeting SMARCA4 (dCas9_SM group) or TFAP2C (dCas9_TF group). As control, a non-injected group was also included. Analysis was carried out in pools of 10 early embryos or 5 blastocysts and at least 3 biological replicates were included. Gene expression was assessed by RTqPCR at Days 2, 4, and 7 after microinjection and data were normalized to that obtained for the non-injected group. The CRISPR-on system was efficient to induce expression of SMARCA4 two days after microinjection (dCas9_SM group, Mann-Whitney t-test; P<0.05), but failed to significantly increase TFAP2C expression (dCas9_TF group). Surprisingly, CDX2, which is a downstream effector for trophectoderm maintenance, was induced both in dCas9_SM and dCas9_TF groups, supporting the CRISPR-mediated induction of targeted transcription factors. However, no changes were observed in the endogenous level of NANOG. Additional analysis is currently ongoing to determine whether CRISPR-on mediated induction of SMARCA4 and/or TFAP2C expression affects lineage specification and regulation. To our knowledge, this is the first report on the use of CRISPR-on for modulation of endogenous gene expression in mammalian embryos. Our study lays the foundations for CRISPR-on application in embryos as a useful tool to understand key cell fate decisions and will enable unprecedented studies of significance to embryo development, cell differentiation, and segregation.

Caged siRNAs with Single cRGD Modification for Photoregulation of Exogenous and Endogenous Gene Expression in Cells and Mice

2018 ◽  
Vol 19 (7) ◽  
pp. 2526-2534 ◽  
Author(s):  
Lijia Yu ◽  
Duanwei Liang ◽  
Changmai Chen ◽  
Xinjing Tang
Keyword(s):  
Gene Expression ◽  
Endogenous Gene ◽  
In Cells

scholarly journals LADL: light-activated dynamic looping for endogenous gene expression control

2019 ◽  
Vol 16 (7) ◽  
pp. 633-639 ◽  
Author(s):  
Ji Hun Kim ◽  
Mayuri Rege ◽  
Jacqueline Valeri ◽  
Margaret C. Dunagin ◽  
Aryeh Metzger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endogenous Gene ◽  
Expression Control ◽  
Gene Expression Control

scholarly journals Protein citrullination as a source of cancer neoantigens

2021 ◽  
Vol 9 (6) ◽  
pp. e002549
Author(s):  
Hiroyuki Katayama ◽  
Makoto Kobayashi ◽  
Ehsan Irajizad ◽  
Alejandro Sevillarno ◽  
Nikul Patel ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Immune Response ◽  
Protein Expression ◽  
Cell Lines ◽  
Cancer Cell ◽  
Family Members ◽  
Immunohistochemical Analysis ◽  
Cancer Cell Lines ◽  
Mhc Ii

BackgroundCitrulline post-translational modification of proteins is mediated by protein arginine deiminase (PADI) family members and has been associated with autoimmune diseases. The role of PADI-citrullinome in immune response in cancer has not been evaluated. We hypothesized that PADI-mediated citrullinome is a source of neoantigens in cancer that induces immune response.MethodsProtein expression of PADI family members was evaluated in 196 cancer cell lines by means of indepth proteomic profiling. Gene expression was assessed using messenger RNA data sets from The Cancer Genome Atlas. Immunohistochemical analysis of PADI2 and peptidyl-citrulline was performed using breast cancer tissue sections. Citrullinated 12–34-mer peptides in the putative Major Histocompatibility Complex-II (MHC-II) binding range were profiled in breast cancer cell lines to investigate the relationship between protein citrullination and antigen presentation. We further evaluated immunoglobulin-bound citrullinome by mass spectrometry using 156 patients with breast cancer and 113 cancer-free controls.ResultsProteomic and gene expression analyses revealed PADI2 to be highly expressed in several cancer types including breast cancer. Immunohistochemical analysis of 422 breast tumor tissues revealed increased expression of PADI2 in ER− tumors (p<0.0001); PADI2 protein expression was positively correlated (p<0.0001) with peptidyl-citrulline staining. PADI2 expression exhibited strong positive correlations with a B cell immune signature and with MHC-II-bound citrullinated peptides. Increased circulating citrullinated antigen–antibody complexes occurred among newly diagnosed breast cancer cases relative to controls (p=0.0012).ConclusionsAn immune response associated with citrullinome is a rich source of neoantigens in breast cancer with a potential for diagnostic and therapeutic applications.

Sign in / Sign up

Export Citation Format

Share Document