scholarly journals Rapid genome editing by CRISPR-Cas9-POLD3 fusion

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Ganna Reint ◽  
Zhuokun Li ◽  
Kornel Labun ◽  
Salla Keskitalo ◽  
Inkeri Soppa ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Dna Sequences ◽  
Gene Editing ◽  
Cell Types ◽  
Cell Type ◽  
Repair Protein ◽  
Specific Manner ◽  
Dna Repair Protein ◽  
Cell Type Specific

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

scholarly journals Rapid genome editing by CRISPR-Cas9-POLD3 fusion

2021 ◽  
Author(s):  
Ganna Reint ◽  
Zhuokun Li ◽  
Kornel Labun ◽  
Salla Keskitalo ◽  
Inkeri Soppa ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Dna Sequences ◽  
Gene Editing ◽  
Cell Types ◽  
Cell Type ◽  
Repair Protein ◽  
Specific Manner ◽  
Dna Repair Protein ◽  
Cell Type Specific

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the particular cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

scholarly journals SeqEnhDL: sequence-based classification of cell type-specific enhancers using deep learning models

2020 ◽  
Author(s):  
Yupeng Wang ◽  
Rosario B. Jaime-Lara ◽  
Abhrarup Roy ◽  
Ying Sun ◽  
Xinyue Liu ◽  
...  
Keyword(s):  
Neural Network ◽  
Deep Learning ◽  
Dna Sequences ◽  
Cell Types ◽  
Learning Models ◽  
Cell Type ◽  
Coding Sequences ◽  
Sequence Features ◽  
Cell Type Specific ◽  
Different Cell Types

AbstractWe propose SeqEnhDL, a deep learning framework for classifying cell type-specific enhancers based on sequence features. DNA sequences of “strong enhancer” chromatin states in nine cell types from the ENCODE project were retrieved to build and test enhancer classifiers. For any DNA sequence, sequential k-mer (k=5, 7, 9 and 11) fold changes relative to randomly selected non-coding sequences were used as features for deep learning models. Three deep learning models were implemented, including multi-layer perceptron (MLP), Convolutional Neural Network (CNN) and Recurrent Neural Network (RNN). All models in SeqEnhDL outperform state-of-the-art enhancer classifiers including gkm-SVM and DanQ, with regard to distinguishing cell type-specific enhancers from randomly selected non-coding sequences. Moreover, SeqEnhDL is able to directly discriminate enhancers from different cell types, which has not been achieved by other enhancer classifiers. Our analysis suggests that both enhancers and their tissue-specificity can be accurately identified according to their sequence features. SeqEnhDL is publicly available at https://github.com/wyp1125/SeqEnhDL.

scholarly journals The complement of desmosomal plaque proteins in different cell types.

1985 ◽  
Vol 101 (4) ◽  
pp. 1442-1454 ◽  
Author(s):  
P Cowin ◽  
H P Kapprell ◽  
W W Franke
Keyword(s):  
Guinea Pig ◽  
Cell Types ◽  
Cardiac Cells ◽  
Myocardial Tissue ◽  
Cell Type ◽  
Wide Range ◽  
Specific Manner ◽  
A Cell ◽  
Cell Type Specific ◽  
Different Cell Types

Desmosomal plaque proteins have been identified in immunoblotting and immunolocalization experiments on a wide range of cell types from several species, using a panel of monoclonal murine antibodies to desmoplakins I and II and a guinea pig antiserum to desmosomal band 5 protein. Specifically, we have taken advantage of the fact that certain antibodies react with both desmoplakins I and II, whereas others react only with desmoplakin I, indicating that desmoplakin I contains unique regions not present on the closely related desmoplakin II. While some of these antibodies recognize epitopes conserved between chick and man, others display a narrow species specificity. The results show that proteins whose size, charge, and biochemical behavior are very similar to those of desmoplakin I and band 5 protein of cow snout epidermis are present in all desmosomes examined. These include examples of simple and pseudostratified epithelia and myocardial tissue, in addition to those of stratified epithelia. In contrast, in immunoblotting experiments, we have detected desmoplakin II only among cells of stratified and pseudostratified epithelial tissues. This suggests that the desmosomal plaque structure varies in its complement of polypeptides in a cell-type specific manner. We conclude that the obligatory desmosomal plaque proteins, desmoplakin I and band 5 protein, are expressed in a coordinate fashion but independently from other differentiation programs of expression such as those specific for either epithelial or cardiac cells.

scholarly journals Quantifying the Tissue-Specific Regulatory Information within Enhancer DNA Sequences

2021 ◽  
Author(s):  
Philipp Benner ◽  
Martin Vingron
Keyword(s):  
Dna Sequences ◽  
Cell Types ◽  
Cell Type ◽  
Regulate Gene Expression ◽  
Tissue Specific ◽  
Sequence Patterns ◽  
Cell Type Specific ◽  
Specific Regulation ◽  
Different Cell Types ◽  
Regulatory Landscape

AbstractRecent efforts to measure epigenetic marks across a wide variety of different cell types and tissues provide insights into the cell type-specific regulatory landscape. We use this data to study if there exists a correlate of epigenetic signals in the DNA sequence of enhancers and explore with computational methods to what degree such sequence patterns can be used to predict cell type-specific regulatory activity. By constructing classifiers that predict in which tissues enhancers are active, we are able to identify sequence features that might be recognized by the cell in order to regulate gene expression. While classification performances vary greatly between tissues, we show examples where our classifiers correctly predict tissue specific regulation from sequence alone. We also show that many of the informative patterns indeed harbor transcription factor footprints.

scholarly journals An inducible genome editing system for plants

2019 ◽  
Author(s):  
Xin Wang ◽  
Lingling Ye ◽  
Robertas Ursache ◽  
Ari Pekka Mähönen
Keyword(s):  
Genome Editing ◽  
Target Gene ◽  
Target Genes ◽  
Biological Process ◽  
Null Alleles ◽  
Cell Type ◽  
Developmental Defects ◽  
Specific Manner ◽  
A Cell ◽  
Cell Type Specific

ABSTRACTConditional manipulation of gene expression is a key approach to investigating the primary function of a gene in a biological process. While conditional and cell-type specific overexpression systems exist for plants, there are currently no systems available to disable a gene completely and conditionally. Here, we present a novel tool with which target genes can be efficiently conditionally knocked out at any developmental stage. The target gene is manipulated using the CRISPR-Cas9 genome editing technology, and conditionality is achieved with the well-established estrogen-inducible XVE system. Target genes can also be knocked-out in a cell-type specific manner. Our tool is easy to construct and will be particularly useful for studying genes which have null-alleles that are non-viable or show strong developmental defects.

scholarly journals A Uve1p-Mediated Mismatch Repair Pathway inSchizosaccharomyces pombe

1999 ◽  
Vol 19 (7) ◽  
pp. 4703-4710 ◽  
Author(s):  
Balveen Kaur ◽  
J. Lee A. Fraser ◽  
Greg A. Freyer ◽  
Scott Davey ◽  
Paul W. Doetsch
Keyword(s):  
Dna Repair ◽  
Mismatch Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Initial Step ◽  
Biochemical Properties ◽  
Repair Pathway ◽  
Repair Protein ◽  
Specific Manner ◽  
Dna Repair Protein

ABSTRACT UV damage endonuclease (Uve1p) from Schizosaccharomyces pombe was initially described as a DNA repair enzyme specific for the repair of UV light-induced photoproducts and proposed as the initial step in an alternative excision repair pathway. Here we present biochemical and genetic evidence demonstrating that Uve1p is also a mismatch repair endonuclease which recognizes and cleaves DNA 5′ to the mispaired base in a strand-specific manner. The biochemical properties of the Uve1p-mediated mismatch endonuclease activity are similar to those of the Uve1p-mediated UV photoproduct endonuclease. Mutants lacking Uve1p display a spontaneous mutator phenotype, further confirming the notion that Uve1p plays a role in mismatch repair. These results suggest that Uve1p has a surprisingly broad substrate specificity and may function as a general type of DNA repair protein with the capacity to initiate mismatch repair in certain organisms.

Abstract 136: Distinct Temporal Requirements for Scl to Repress Cardiomyogenesis in Endothelial-derived Cells in Hemogenic Tissues and the Heart

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Ben Van Handel ◽  
Tonis Org ◽  
Amelie Montel-Hagen ◽  
Haruko Nakano ◽  
Atsushi Nakano ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Time Window ◽  
Ectopic Expression ◽  
Cell Types ◽  
Yolk Sac ◽  
Lineage Tracing ◽  
Cell Type ◽  
Specific Manner ◽  
A Cell ◽  
Cell Type Specific

Identification of precursors with the capacity to generate cardiomyocytes is critical for advancing cardiac regenerative medicine. By analyzing knockout embryos for the bHLH factor Scl, we demonstrated that endothelial cells in hematopoietic tissues and the heart possess latent cardiomyogenic capacity. Furthermore, analysis of tamoxifen-inducible Rosa26-Cre ERT2 Scl fl/fl embryos suggested that the time window during which Scl is required for cardiac repression extends later in the heart versus the yolk sac. However, the cell types in which Scl acts remained elusive. We then deleted Scl in a cell-type specific manner in early mesoderm using Mesp1-Cre and in endothelial cells using Tie2-Cre. Lineage tracing in Mesp1-Cre Rosa26-YFP embryos demonstrated that at E9.5, a large majority of hematopoietic and endothelial cells in the yolk sac and heart were labeled. Moreover, deletion of Scl in Mesp1-Cre Scl fl/fl embryos phenocopied the germline knockout, essentially abrogating hematopoiesis and promoting the emergence of CD31 + PDGFRα + cardiomyogenic precursors and ectopic expression of the cardiomyocyte genes Myl7 and Tnnt2 in yolk sac vasculature. In contrast, deletion of Scl after endothelium had been specified in Tie2-Cre Scl fl/fl embryos did not grossly affect yolk sac hematopoiesis, nor did it induce ectopic cardiomyogenesis in hemogenic tissues. However, endothelial-derived cells in the hearts of Tie2-Cre Scl fl/fl embryos evidenced profound expansion of CD31 + PDGFRα + cardiogenic precursors at E11.5 and E13.5, as well as displayed dramatic upregulation of Myl7 and Tnnt2 , showing that the requirement for Scl to repress the cardiomyogenic program extends longer in endothelial derivatives in the heart than in the yolk sac. These data demonstrate that endocardial-derived cells in the heart retain latent cardiomyogenic potential until mid-gestation and nominate Scl as a critical regulator of endocardial fate.

scholarly journals A Knockout Mouse Approach Reveals that TCTP Functions as an Essential Factor for Cell Proliferation and Survival in a Tissue- or Cell Type–specific Manner

2007 ◽  
Vol 18 (7) ◽  
pp. 2525-2532 ◽  
Author(s):  
Sung Ho Chen ◽  
Peih-Shan Wu ◽  
Chiang-Hung Chou ◽  
Yu-Ting Yan ◽  
Hsuan Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Types ◽  
Cell Type ◽  
Cellular Functions ◽  
Embryonic Fibroblasts ◽  
Embryonic Tissues ◽  
Targeted Gene Disruption ◽  
Specific Manner ◽  
Cell Type Specific ◽  
And Control

Translationally controlled Tumor Protein (TCTP) is an evolutionally highly conserved protein which has been implicated in many cellular functions that are related to cell growth, death, and even the allergic response of the host. To address the physiological roles of TCTP, we generated TCTP knockout mice by targeted gene disruption. Heterozygous mutants appeared to be developmentally normal. However, homozygous mutants (TCTP−/−) were embryonic lethal. TCTP−/− embryos were smaller in size than the control littermates at all postimplantation stages examined. Although TCTP is widely expressed in both extraembryonic and embryonic tissues, the most prominent defect of the TCTP−/− embryo at embryonic stage day 5.5 (E5.5) was in its epiblast, which had a reduced number of cells compared with wild-type controls. The knockout embryos also suffered a higher incidence of apoptosis in epiblast starting about E6.5 and subsequently died around E9.5–10.5 with a severely disorganized structure. Last, we demonstrated that TCTP−/− and control mouse embryonic fibroblasts manifested similar proliferation activities and apoptotic sensitivities to various death stimuli. Taken together, our results suggest that despite that TCTP is widely expressed in many tissues or cell types, it appears to regulate cell proliferation and survival in a tissue- or cell type–specific manner.

scholarly journals Haystack: systematic analysis of the variation of epigenetic states and cell-type specific regulatory elements

2017 ◽  
Author(s):  
Luca Pinello ◽  
Rick Farouni ◽  
Guo-Cheng Yuan
Keyword(s):  
Dna Sequences ◽  
Cell Types ◽  
Regulatory Elements ◽  
Epigenetic Mark ◽  
Cell Type ◽  
Epigenetic Variability ◽  
Bioinformatics Pipeline ◽  
Systematic Analysis ◽  
Regulatory Dna Sequences ◽  
Cell Type Specific

AbstractMotivationWith the increasing amount of genomic and epigenomic data in the public domain, a pressing challenge is how to integrate these data to investigate the role of epigenetic mechanisms in regulating gene expression and maintenance of cell-identity. To this end, we have implemented a computational pipeline to systematically study epigenetic variability and uncover regulatory DNA sequences that play a role in gene regulation.ResultsHaystack is a bioinformatics pipeline to characterize hotspots of epigenetic variability across different cell-types as well as cell-type specific cis-regulatory elements along with their corresponding transcription factors. Our approach is generally applicable to any epigenetic mark and provides an important tool to investigate cell-type identity and the mechanisms underlying epigenetic switches during development. Additionally, we make available a set of precomputed tracks for a number of epigenetic marks across several cell types. These precomputed results may be used as an independent resource for functional annotation of the human genome.AvailabilityThe Haystack pipeline is implemented as an open-source, multiplatform, Python package called haystack_bio available at https://github.com/pinellolab/[email protected], [email protected]

scholarly journals Quantifying the tissue-specific regulatory information within enhancer DNA sequences

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Philipp Benner ◽  
Martin Vingron
Keyword(s):  
Dna Sequences ◽  
Cell Types ◽  
Cell Type ◽  
Regulate Gene Expression ◽  
Tissue Specific ◽  
Sequence Patterns ◽  
Cell Type Specific ◽  
Specific Regulation ◽  
Different Cell Types ◽  
Regulatory Landscape

Abstract Recent efforts to measure epigenetic marks across a wide variety of different cell types and tissues provide insights into the cell type-specific regulatory landscape. We use these data to study whether there exists a correlate of epigenetic signals in the DNA sequence of enhancers and explore with computational methods to what degree such sequence patterns can be used to predict cell type-specific regulatory activity. By constructing classifiers that predict in which tissues enhancers are active, we are able to identify sequence features that might be recognized by the cell in order to regulate gene expression. While classification performances vary greatly between tissues, we show examples where our classifiers correctly predict tissue-specific regulation from sequence alone. We also show that many of the informative patterns indeed harbor transcription factor footprints.

Sign in / Sign up

Export Citation Format

Share Document