scholarly journals Engineering improved Cas13 effectors for targeted post-transcriptional regulation of gene expression

2021 ◽  
Author(s):  
Emeric J Charles ◽  
Shin Eui Kim ◽  
Gavin J Knott ◽  
Dylan Smock ◽  
Jennifer Doudna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Directed Evolution ◽  
Regulation Of Gene Expression ◽  
Exon Skipping ◽  
Cell Types ◽  
Human Cells ◽  
Translational Repression ◽  
E Coli ◽  
Rna Targeting

Cas13 is a family of unique RNA-targeting CRISPR-Cas effectors, making it an appealing tool for probing and perturbing RNA function. However only a few Cas13 homologs have been shown to mediate robust RNA targeting in human cells, suggesting that unknown elements may be limiting their efficacy. Furthermore, many Cas13 enzymes show high degrees of toxicity upon targeting and have not been shown to mediate specific knockdown in other cell types such as E. coli. Here, we show that catalytically inactive Cas13 enzymes can be repurposed for efficient translational repression in bacteria with no associated growth defects. To achieve this advance, we carried out a directed evolution screen to engineer functional Cas13a variants, and identified a number of stabilizing mutations, which enabled efficient post transcriptional knockdown of gene expression. In vitro characterization of the resulting engineered Lbu Cas13a mutant, termed eLbu, revealed both stabilization and altered cleavage kinetics. Finally, we show that eLbu can be used for efficient exon skipping in human cells. This work represents the first demonstration of targeted translational repression in E. coli using a CRISPR enzyme, as well as the first directed evolution of a Cas13a enzyme. Such a platform could allow for engineering other aspects of this protein family to obtain more robust RNA targeting tools.

scholarly journals TET-catalyzed 5-hydroxymethylcytosine regulates gene expression in differentiating colonocytes and colon cancer

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Christopher G. Chapman ◽  
Christopher J. Mariani ◽  
Feng Wu ◽  
Katherine Meckel ◽  
Fatma Butun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colon Cancer ◽  
Regulation Of Gene Expression ◽  
Human Colon ◽  
Cell Types ◽  
Enzymatic Oxidation ◽  
Dna Hydroxymethylation ◽  
Differentiated Cells ◽  
Colon Cancers

Abstract The formation of differentiated cell types from pluripotent progenitors involves epigenetic regulation of gene expression. DNA hydroxymethylation results from the enzymatic oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (TET) 5-mC dioxygenase enzymes. Previous work has mapped changes in 5-mC during differentiation of intestinal stem cells. However, whether or not 5-hmC regulates colonocyte differentiation is unknown. Here we show that 5-hmC regulates gene expression during colonocyte differentiation and controls gene expression in human colon cancers. Genome-wide profiling of 5-hmC during in vitro colonic differentiation demonstrated that 5-hmC is gained at highly expressed and induced genes and is associated with intestinal transcription factor binding sites, including those for HNF4A and CDX2. TET1 induction occurred during differentiation and TET1 knockdown altered gene expression and inhibited barrier formation of colonocytes. We find that the 5-hmC distribution in primary human colonocytes parallels the distribution found in differentiated cells in vitro and that gene-specific 5-hmC changes in human colon cancers are directly correlated with changes in gene expression. Our results support a model in which 5-hmC regulates differentiation of adult human intestine and 5-hmC alterations contribute to the disrupted gene expression in colon cancer.

scholarly journals PSIV-5 Developmental and hormonal regulation of gene expression of fibrillin-1 (FBN1) and the asprosin receptor, olfactory receptor family 4 subfamily M member 1 (OR4M1), in bovine ovarian cells

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 283-284
Author(s):  
Excel Rio Maylem ◽  
Leon Spicer ◽  
Isadora Batalha ◽  
Luis Schutz
Keyword(s):  
Gene Expression ◽  
Hormonal Regulation ◽  
Regulation Of Gene Expression ◽  
Cell Types ◽  
Mrna Abundance ◽  
Hormone Regulation ◽  
Polycystic Ovaries ◽  
Fbn1 Gene ◽  
Fibrillin 1

Abstract Asprosin is a novel fasting-induced protein associated with insulin resistance and polycystic ovaries in humans. It is encoded by FBN1 gene and produced when FBN1 is cleaved by the enzyme furin. In cattle, the role of asprosin is unknown. To characterize mRNA abundance of FBN1, furin, and the asprosin receptor, OR4M1, in granulosa (GC) and theca cells (TC), and identify hormones regulating FBN1 mRNA expression, GC and TC from small (< 6 mm; SM) and large (>5 mm; LG) follicles were collected from heifers at an abattoir and used for real-time gene expression analysis or in vitro evaluation of hormone regulation. SMTC had 151-fold greater (P < 0.05) FBN1 mRNA abundance than SMGC, and LGTC had 50-fold greater (P < 0.05) FBN1 mRNA than LGGC. In contrast, OR4M1 mRNA abundance was significantly greater (by 81-fold) in SMGC than LGGC and did not differ from SMTC, but LGTC had 9-fold greater (P < 0.05) OR4M1 mRNA abundance than LGGC. Furin mRNA was significantly greater (by 2.6-fold) in SMGC than SMTC but did not differ between LGTC and LGGC. In SMGC, leptin, insulin, GH, FSH, EGF, and steroids had no effect (P >0.10) on FBN1 mRNA abundance. In contrast, TGFB1, WNT3A and FGF9 increased (P < 0.05) and IGF1 significantly decreased SMGC FBN1 mRNA abundance. In LGTC, leptin, insulin, LH, IGF1 and steroids did not significantly affect FBN1 mRNA, but TGFB1, WNT3A, EGF, FGF2 and FGF9 increased (P< 0.05) FBN1 mRNA abundance. Altogether, FBN1 mRNA was more highly expressed in TC than GC and was stimulated by TGFB1, WNT3A and FGF9 in both cell types. Developmental and hormonal regulation of FBN1, furin and OR4M1 along with a greater expression of OR4M1 mRNA in GC than TC suggests that asprosin may be acting as a paracrine regulator of ovarian follicular function in cattle.

Control of Transcription of tRNA Genes

1981 ◽  
Vol 39 ◽  
pp. 480-481
Author(s):  
K.U. Sprague ◽  
D. Morton ◽  
D. Larson
Keyword(s):  
Gene Expression ◽  
Bombyx Mori ◽  
Genomic Dna ◽  
Molecular Mechanisms ◽  
Regulation Of Gene Expression ◽  
Cell Types ◽  
The Other ◽  
Trna Genes ◽  
Trna Species

Our laboratory is interested in the regulation of gene expression during eukaryotic development. Among the genes we are currently studying are those coding for alanine tRNA in the silkworm, Bombyx mori. These genes are particularly interesting because one Bombyx alanine tRNA species is constitutive -- that is, it is found in a variety of cell types -- while the other major alanine tRNA appears only in the silkgland. The silkgland-specific tRNA predominates when the highly specialized cells of this tissue are synthesizing large amounts of the alanine-rich silk protein, fibroin. The nucleotide sequences of the two alanine tRNAs indicates that they are encoded by different genes.Our goal is to understand the molecular mechanisms governing the differential expression of Bombyx tRNAAla genes. Our approach is to isolate the genes with molecular cloning techniques, and to transcribe and process them in vitro. We have cloned 14 different genomic DNA fragments containing tRNAAla genes, and have developed faithful transcription systems from several different Bombyx tissues, including silkgland.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals Fos and jun cooperate in transcriptional regulation via heterologous activation domains.

1990 ◽  
Vol 10 (10) ◽  
pp. 5532-5535 ◽  
Author(s):  
C Abate ◽  
D Luk ◽  
E Gagne ◽  
R G Roeder ◽  
T Curran
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Regulation Of Gene Expression ◽  
Negative Influence ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Transcriptional Stimulation

The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site. Here we have examined the contribution of Fos and Jun toward transcriptional activity by using Fos and Jun polypeptides purified from Escherichia coli. Fos contained a transcriptional activation domain as well as a region which exerted a negative influence on transcriptional activity in vitro. Moreover, distinct activation domains in both Fos and Jun functioned cooperatively in transcriptional stimulation. Thus, regulation of gene expression by Fos and Jun results from an integration of several functional domains in a bimolecular complex.

scholarly journals Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM

2019 ◽  
Author(s):  
Marcus Alvarez ◽  
Elior Rahmani ◽  
Brandon Jew ◽  
Kristina M. Garske ◽  
Zong Miao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Types ◽  
Supervised Machine Learning ◽  
Data Sets ◽  
Rna Seq ◽  
Novel Approach ◽  
Single Nucleus ◽  
Downstream Analysis

AbstractSingle-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. Contrary to single-cell RNA seq (scRNA-seq), we observe that snRNA-seq is commonly subject to contamination by high amounts of extranuclear background RNA, which can lead to identification of spurious cell types in downstream clustering analyses if overlooked. We present a novel approach to remove debris-contaminated droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: 1) human differentiating preadipocytes in vitro, 2) fresh mouse brain tissue, and 3) human frozen adipose tissue (AT) from six individuals. All three data sets showed various degrees of extranuclear RNA contamination. We observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq data, we also successfully applied DIEM to single-cell data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

scholarly journals Cell-type Specific Expression Quantitative Trait Loci Associated with Alzheimer Disease in Blood and Brain Tissue

2020 ◽  
Author(s):  
Devanshi Patel ◽  
Xiaoling Zhang ◽  
John J. Farrell ◽  
Jaeyoon Chung ◽  
Thor D. Stein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Trait ◽  
Expression Patterns ◽  
Regulation Of Gene Expression ◽  
Cell Types ◽  
Eqtl Analysis ◽  
Cell Type ◽  
Specific Expression ◽  
Cell Type Specific Expression ◽  
Cell Type Specific

ABSTRACTBecause regulation of gene expression is heritable and context-dependent, we investigated AD-related gene expression patterns in cell-types in blood and brain. Cis-expression quantitative trait locus (eQTL) mapping was performed genome-wide in blood from 5,257 Framingham Heart Study (FHS) participants and in brain donated by 475 Religious Orders Study/Memory & Aging Project (ROSMAP) participants. The association of gene expression with genotypes for all cis SNPs within 1Mb of genes was evaluated using linear regression models for unrelated subjects and linear mixed models for related subjects. Cell type-specific eQTL (ct-eQTL) models included an interaction term for expression of “proxy” genes that discriminate particular cell type. Ct-eQTL analysis identified 11,649 and 2,533 additional significant gene-SNP eQTL pairs in brain and blood, respectively, that were not detected in generic eQTL analysis. Of note, 386 unique target eGenes of significant eQTLs shared between blood and brain were enriched in apoptosis and Wnt signaling pathways. Five of these shared genes are established AD loci. The potential importance and relevance to AD of significant results in myeloid cell-types is supported by the observation that a large portion of GWS ct-eQTLs map within 1Mb of established AD loci and 58% (23/40) of the most significant eGenes in these eQTLs have previously been implicated in AD. This study identified cell-type specific expression patterns for established and potentially novel AD genes, found additional evidence for the role of myeloid cells in AD risk, and discovered potential novel blood and brain AD biomarkers that highlight the importance of cell-type specific analysis.

scholarly journals Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

2012 ◽  
Vol 8 (6) ◽  
pp. e1002728 ◽  
Author(s):  
Hermine Mohr ◽  
Christian A. Mohr ◽  
Marlon R. Schneider ◽  
Laura Scrivano ◽  
Barbara Adler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression

scholarly journals Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Ana J. Chucair-Elliott ◽  
Sarah R. Ocañas ◽  
David R. Stanford ◽  
Victor A. Ansere ◽  
Kyla B. Buettner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Cell Types ◽  
Tissue Level ◽  
Cell Phenotype ◽  
Specific Gene ◽  
Cell Type ◽  
A Cell ◽  
Cell Type Specific

AbstractEpigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific central nervous system cell types. Analysis of gene expression and DNA modifications in astrocytes or microglia from the same animal demonstrates differential usage of DNA methylation and hydroxymethylation in CpG and non-CpG contexts that corresponds to cell type-specific gene expression. Application of this approach in LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model and the validation approaches presented can be applied to any brain cell type for which a cell type-specific cre is available.

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