scholarly journals Unintended inhibition of protein function using GFP nanobodies in human cells

2019 ◽  
Author(s):  
Cansu Küey ◽  
Gabrielle Larocque ◽  
Nicholas I. Clarke ◽  
Stephen J. Royle
Keyword(s):  
Developmental Biology ◽  
Genome Editing ◽  
Cell Lines ◽  
Protein Function ◽  
Human Cells ◽  
Popular Approach ◽  
Inactive State ◽  
Dynamin 2 ◽  
Gfp Tag

Tagging a protein-of-interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed “dongles”, that could add, for example, an FKBP tag to a GFP-tagged protein-of-interest; enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2-GFP caused inhibition of dynamin function prior to knocksideways. While this limitation might be specific to the protein studied, it was significant enough to convince us not to pursue development of dongle technology further.

scholarly journals piggyPrime: High-Efficacy Prime Editing in Human Cells Using piggyBac-Based DNA Transposition

2021 ◽  
Vol 3 ◽  
Author(s):  
Jonas Holst Wolff ◽  
Jakob Haldrup ◽  
Emil Aagaard Thomsen ◽  
Sofie Andersen ◽  
Jacob Giehm Mikkelsen
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Cell Lines ◽  
Human Cells ◽  
Vector System ◽  
High Efficacy ◽  
Genomic Alterations ◽  
Dna Transposition ◽  
Genomic Integration ◽  
Wide Range

Prime editing is a novel genome editing technology that allows a wide range of tailored genomic alterations. Prime editing does not involve homologous recombination, but suffers from low efficacy. Here, we demonstrate piggyPrime, a transfected single-vector system based on piggyBac DNA transposition for genomic integration of all prime editing components in human cells allowing easy and effective transgenesis with prime editing efficacies up to 100% in cell lines.

scholarly journals Generation of Double-Labeled Reporter Cell Lines for Studying Co-Dynamics of Endogenous Proteins in Individual Human Cells

PLoS ONE ◽  
2010 ◽  
Vol 5 (10) ◽  
pp. e13524 ◽  
Author(s):  
Irina Issaeva ◽  
Ariel A. Cohen ◽  
Eran Eden ◽  
Cellina Cohen-Saidon ◽  
Tamar Danon ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cells ◽  
Reporter Cell ◽  
Endogenous Proteins ◽  
Individual Human

CRISPR-Cpf1-mediated genome editing and gene regulation in human cells

2019 ◽  
Vol 37 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Tianwen Li ◽  
Linwen Zhu ◽  
Bingxiu Xiao ◽  
Zhaohui Gong ◽  
Qi Liao ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Genome Editing ◽  
Human Cells

scholarly journals Transcriptomic Analysis of the Differential Nephrotoxicity of Diverse Brominated Flame Retardants in Rat and Human Renal Cells

2021 ◽  
Vol 22 (18) ◽  
pp. 10044
Author(s):  
Lillie Marie A. Barnett ◽  
Naomi E. Kramer ◽  
Amanda N. Buerger ◽  
Deirdre H. Love ◽  
Joseph H. Bisesi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Flame Retardants ◽  
Molecular Mechanisms ◽  
Annexin V ◽  
Enrichment Analysis ◽  
Brominated Flame Retardants ◽  
Human Cells ◽  
Gene Set Enrichment Analysis ◽  
Coagulation Cascade ◽  
Transcriptional Changes

Brominated flame retardants (BFRs) are environmentally persistent, are detected in humans, and some have been banned due to their potential toxicity. BFRs are developmental neurotoxicants and endocrine disruptors; however, few studies have explored their potential nephrotoxicity. We addressed this gap in the literature by determining the toxicity of three different BFRs (tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD), and tetrabromodiphenyl ether (BDE-47)) in rat (NRK 52E) and human (HK-2 and RPTEC) tubular epithelial cells. All compounds induced time- and concentration-dependent toxicity based on decreases in MTT staining and changes in cell and nuclear morphology. The toxicity of BFRs was chemical- and cell-dependent, and human cells were more susceptible to all three BFRs based on IC50s after 48 h exposure. BFRs also had chemical- and cell-dependent effects on apoptosis as measured by increases in annexin V and PI staining. The molecular mechanisms mediating this toxicity were investigated using RNA sequencing. Principal components analysis supported the hypothesis that BFRs induce different transcriptional changes in rat and human cells. Furthermore, BFRs only shared nine differentially expressed genes in rat cells and five in human cells. Gene set enrichment analysis demonstrated chemical- and cell-dependent effects; however, some commonalities were also observed. Namely, gene sets associated with extracellular matrix turnover, the coagulation cascade, and the SNS-related adrenal cortex response were enriched across all cell lines and BFR treatments. Taken together, these data support the hypothesis that BFRs induce differential toxicity in rat and human renal cell lines that is mediated by differential changes in gene expression.

scholarly journals CRISPR-Cas9 Genome Editing in Human Cell Lines with Donor Vector Made by Gibson Assembly

2020 ◽  
pp. 365-383
Author(s):  
Nirakar Sahoo ◽  
Victoria Cuello ◽  
Shreya Udawant ◽  
Carl Litif ◽  
Julie A. Mustard ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Lines ◽  
Human Cell ◽  
Human Cell Lines ◽  
Gibson Assembly ◽  
Donor Vector

scholarly journals Efficient target cleavage by Type V Cas12a effector programmed with split CRISPR RNA

2020 ◽  
Author(s):  
Regina Tkach ◽  
Natalia Nikitchina ◽  
Nikita Shebanov ◽  
Vladimir Mekler ◽  
Egor Ulashchik ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Target Recognition ◽  
Human Cells ◽  
Stable Complex ◽  
Slight Effect ◽  
Target Dna ◽  
Type V

ABSTRACTCRISPR RNAs (crRNAs) directing target DNA cleavage by type V-A Cas12a nucleases consist of repeat-derived 5’-scaffold moiety and 3’-spacer moiety. We demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by Cas12a ortholog from Acidaminococcus sp (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer part only, while crRNAs split into two individual moieties (scaffold and spacer RNAs) catalyzed highly specific and efficient cleavage of target DNA. Our data also indicate that AsCas12a combined with split crRNA forms a stable complex with the target. These observations were also confirmed in lysates of human cells expressing AsCas12a. The ability of the AsCas12a nuclease to be programmed with split crRNAs opens new lines of inquiry into the mechanisms of target recognition and cleavage and will further facilitate genome editing techniques based on Cas12a nucleases.

scholarly journals Efficient genome editing in multiple salmonid cell lines using ribonucleoprotein complexes

2020 ◽  
Author(s):  
Remi L. Gratacap ◽  
Ye Hwa Jin ◽  
Marina Mantsopoulou ◽  
Ross D. Houston
Keyword(s):  
Rainbow Trout ◽  
Atlantic Salmon ◽  
Infectious Diseases ◽  
Genome Editing ◽  
Cell Lines ◽  
Chinook Salmon ◽  
Model Systems ◽  
Fish Cell ◽  
Ribonucleoprotein Complexes ◽  
Salmonid Species

AbstractInfectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide a valuable model to study infectious diseases in salmonids, and genome editing using CRISPR provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas9 has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly-used salmonid fish cell lines; Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90 % cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture.

scholarly journals Repair of a previously uncharacterized second host-range gene contributes to full replication of modified vaccinia virus Ankara (MVA) in human cells

2020 ◽  
Vol 117 (7) ◽  
pp. 3759-3767 ◽  
Author(s):  
Chen Peng ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Host Range ◽  
Cell Lines ◽  
Chicken Embryo ◽  
A549 Cells ◽  
Human Cells ◽  
Modified Vaccinia Virus Ankara ◽  
Core Proteins ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Modified vaccinia virus Ankara (MVA), a widely used vaccine vector for expression of genes of unrelated pathogens, is safe, immunogenic, and can incorporate large amounts of added DNA. MVA was derived by extensively passaging the chorioallantois vaccinia virus Ankara (CVA) vaccine strain in chicken embryo fibroblasts during which numerous mutations and deletions occurred with loss of replicative ability in most mammalian cells. Restoration of the deleted C12L gene, encoding serine protease inhibitor 1, enhances replication of MVA in human MRC-5 cells but only slightly in other human cells. Here we show that repair of the inactivated C16L/B22R gene of MVA enhances replication in numerous human cell lines. This previously uncharacterized gene is present at both ends of the genome of many orthopoxviruses and is highly conserved in most, including smallpox and monkeypox viruses. The C16L/B22R gene is expressed early in infection from the second methionine of the previously annotated Copenhagen strain open reading frame (ORF) as a 17.4-kDa protein. The C16/B22 and C12 proteins together promote MVA replication in human cells to levels that are comparable to titers in chicken embryo fibroblasts. Both proteins enhance virion assembly, but C16/B22 increases proteolytic processing of core proteins in A549 cells consistent with higher infectious virus titers. Furthermore, human A549 cells expressing codon-optimized C16L/B22R and C12L genes support higher levels of MVA replication than cell lines expressing neither or either alone. Identification of the genes responsible for the host-range defect of MVA may allow more rational engineering of vaccines for efficacy, safety, and propagation.

scholarly journals Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Abdullah O. Khan ◽  
Carl W. White ◽  
Jeremy A. Pike ◽  
Jack Yule ◽  
Alexandre Slater ◽  
...  
Keyword(s):  
Genome Editing ◽  
G Protein ◽  
Single Molecule ◽  
Protein Function ◽  
Single Molecule Imaging ◽  
Dependent Effect ◽  
Systematic Comparison ◽  
Selection Strategies ◽  
Localisation Microscopy ◽  
G Protein Coupled

Abstract The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.

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