Ontology highlight
ABSTRACT:
PROVIDER: PRJNA388126 | ENA |
REPOSITORIES: ENA
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Project description:The targeting range of CRISPR-Cas9 base editors (BEs) is limited by their G/C-rich PAM sequences. To overcome this limitation, we developed a CRISPR/Cpf1-based BE by fusing the rat cytosine deaminase APOBEC1 to a catalytically inactive version of Lachnospiraceae bacterium Cpf1. The base editor recognizes a T-rich PAM sequence and converts C to T in human cells with low levels of indels, non-C-to-T substitutions and off-target editing.
2018-03-18 | GSE110136 | GEO
Project description:C-to-T base editing mediated by CRISPR/Cas9 base editors (BEs) needs a G/C-rich PAM and the editing fidelity is compromised by unwanted indels and non-C-to-T substitutions. We developed CRISPR/Cpf1-based BEs to recognize a T-rich PAM and induce efficient C-to-T editing with few indels and/or non-C-to-T substitutions. The requirement of editing fidelity in therapeutic-related trials necessitates the development of CRISPR/Cpf1-based BEs, which also facilitates base editing in A/T-rich regions.
2018-10-16 | GSE105002 | GEO
Project description:Nuclear receptors function as ligand-regulated transcription factors whose ability to regulate diverse physiological processes is closely linked with conformational changes induced upon ligand binding. Understanding how conformational populations of nuclear receptors are shifted by various ligands could illuminate strategies for the design of synthetic modulators to regulate specific transcriptional programs. Here, we investigate ligand-induced conformational changes using a reconstructed, ancestral nuclear receptor. By making substitutions at a key position, we engineer receptor variants with altered ligand specificities. We use atomistic molecular dynamics (MD) simulations with enhanced sampling to generate ensembles of wildtype and engineered receptors in combination with multiple ligands, followed by conformational analysis and prediction of ligand activity. We combine cellular and biophysical experiments to allow correlation of MD-based predictions with functional ligand profiles, as well as elucidation of mechanisms underlying altered transcription in receptor variants. We determine that conformational ensembles accurately predict ligand responses based on observed population shifts, even within engineered receptors that were constitutively active or transcriptionally unresponsive in experiments. These studies provide a platform which will allow structural characterization of physiologically-relevant conformational ensembles, as well as provide the ability to design and predict transcriptional responses in novel ligands.
2022-10-12 | PXD036076 | Pride
Project description:A key limitation of the commonly-used CRISPR enzyme S. pyogenes Cas9 is the strict requirement of an NGG protospacer-adjacent motif (PAM) at the target site, which reduces the number of accessible genomic loci. This constraint can be limiting for genome editing applications that require precise Cas9 positioning. Recently, two Cas9 variants with a relaxed PAM requirement (NG) have been developed (xCas9 and Cas9-NG) but their activity has been measured at only a small number of endogenous sites. Here we devised a high-throughput Cas9 pooled competition screen to compare the performance of both PAM-flexible Cas9 variants and wild-type Cas9 at thousands of genomic loci and across 3 modalities (gene knock-out, transcriptional activation and suppression). We show that PAM flexibility comes at a substantial cost of decreased DNA targeting and cutting. Of the PAM-flexible variants, we found that Cas9-NG outperforms xCas9 regardless of genome engineering modality or PAM. Finally, we combined xCas9 mutations with those of Cas9-NG, creating a stronger transcriptional modulator than existing PAM-flexible Cas9 variants.
2020-01-26 | GSE143892 | GEO
Project description:CRISPR-Cas transcriptional tools have been widely applied for programmable regulation of complex biological networks. In comparison to eukaryotic systems, bacterial CRISPR activation (CRISPRa) has stringent target site requirements for effective gene activation. While genes may not always have an NGG protospacer adjacent motif (PAM) at the appropriate position, PAM-flexible dCas9 variants can expand the range of targetable sites. Here we systematically evaluate a panel of PAM-flexible dCas9 variants for their ability to activate bacterial genes. We observe that dxCas9-NG provides a high dynamic range of gene activation for sites with NGN PAMs while dSpRY permits modest activity across almost any PAM. Similar trends were observed for heterologous and endogenous promoters. For all variants tested, improved PAM-flexibility comes with the tradeoff that CRISPRi-mediated gene repression becomes less effective. Weaker CRISPR interference (CRISPRi) gene repression can be partially rescued by expressing multiple sgRNAs to target many sites in the gene of interest. Our work provides a framework to choose the most effective dCas9 variant for a given set of gene targets, which will further expand the utility of CRISPRa/i gene regulation in bacterial systems.
2022-11-15 | GSE217559 | GEO
Project description:Human somatic cells may contain up to seven members of the histone H1 family contributing to chromatin compaction and regulation of nuclear processes, apparently with certain subtype specificities. Previous studies in T47D breast cancer cells determined that H1 variants are distributed in a variant-specific manner throughout the genome, although only H1.2 and H1X endogenous variants were mapped. Now, we performed ChIP-seq of five endogenous H1 variants (H1.0, H1.2, H1.4, H1.5, H1X) in T47D cells.
2021-08-16 | GSE166645 | GEO
Project description:There is an absolute requirement of Pax7 for the normal function of MuSCs during regenerative myogenesis in skeletal muscle at any stage of life. Here using RNA-seq, H3K27ac and Pax7 ChIP-seq, we discover PAM-1 (Pax7 Associated Muscle lncRNA) that is enriched in activated skeletal muscle satellite cells (ASCs) 24 and 48 hours after activation. Knockdown of PAM-1 reduces proliferating Pax7+Myod+ ASCs number, while overexpression of PAM-1 increases ASCs number. Mechanistically, PAM-1 is located on ASCs and myoblast specific super-enhancer (SE), and we categorize it as seRNA. Through a series of multiomics analysis of PAM-1 interactome in myoblast including PAM-1-DNA interaction by ChIRP-seq, PAM-1 SE-DNA interaction by 4C-seq, PAM-1-protein interaction by mass spectrometry and ChIP-seq, we identify a novel class of transcriptional regulation that seRNA PAM-1 interacts with RNA binding protein Ddx5 and tethers PAM-1 SE to regulate inter-chromosomal targets Timp2. Altogether, our findings identify PAM-1 is driven by Pax7 in ASC and myoblast to regulate myogenic activation through binding with Ddx5 and targeting Timp2.
2022-07-12 | GSE180071 | GEO
Project description:There is an absolute requirement of Pax7 for the normal function of MuSCs during regenerative myogenesis in skeletal muscle at any stage of life. Here using RNA-seq, H3K27ac and Pax7 ChIP-seq, we discover PAM-1 (Pax7 Associated Muscle lncRNA) that is enriched in activated skeletal muscle satellite cells (ASCs) 24 and 48 hours after activation. Knockdown of PAM-1 reduces proliferating Pax7+Myod+ ASCs number, while overexpression of PAM-1 increases ASCs number. Mechanistically, PAM-1 is located on ASCs and myoblast specific super-enhancer (SE), and we categorize it as seRNA. Through a series of multiomics analysis of PAM-1 interactome in myoblast including PAM-1-DNA interaction by ChIRP-seq, PAM-1 SE-DNA interaction by 4C-seq, PAM-1-protein interaction by mass spectrometry and ChIP-seq, we identify a novel class of transcriptional regulation that seRNA PAM-1 interacts with RNA binding protein Ddx5 and tethers PAM-1 SE to regulate inter-chromosomal targets Timp2. Altogether, our findings identify PAM-1 is driven by Pax7 in ASC and myoblast to regulate myogenic activation through binding with Ddx5 and targeting Timp2.
2022-07-12 | GSE180070 | GEO