Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination

Abstract Background Precise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathway and depends on the presence of repair templates its efficiency is often limited and demands optimized methodology. Results For the enhancement of HDR we redirect the DSB repair pathway choice by targeting the Ubiquitin mark for damaged chromatin at Histone H2A-K15. We used fusions of the Ubiquitin binding domain (UBD) of Rad18 or RNF169 with BRCA1 to promote HDR initiation and UBD fusions with DNA binding domains to attract donor templates and facilitate HDR processing. Using a traffic light reporter system in human HEK293 cells we found that the coexpression of both types of UBD fusion proteins promotes HDR, reduces NHEJ and shifts the HDR/NHEJ balance up to 6-fold. The HDR enhancing effect of UBD fusion proteins was confirmed at multiple endogenous loci. Conclusions Our findings provide a novel efficient approach to promote precise gene editing in human cells.

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Homology Directed Repair by Cas9:Donor Co-localization in Mammalian Cells

10.1101/248179 ◽

2018 ◽

Author(s):

Philip J.R. Roche ◽

Heidi Gytz ◽

Faiz Hussain ◽

Christopher J.F. Cameron ◽

Denis Paquette ◽

...

Keyword(s):

Mammalian Cells ◽

Gene Editing ◽

Low Frequency ◽

Cost Effective ◽

Hek293 Cells ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Homology Directed Repair ◽

Non Homologous End Joining

AbstractHomology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.SummaryPrecision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence.

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Spatial and temporal control of CRISPR/Cas9-mediated gene editing delivered via a light-triggered liposome system

10.1101/725465 ◽

2019 ◽

Author(s):

Yagiz Alp Aksoy ◽

Wenjie Chen ◽

Ewa M Goldys ◽

Wei Deng

Keyword(s):

Gene Editing ◽

Transfection Efficiency ◽

Hek293 Cells ◽

Temporal Control ◽

Reporter System ◽

Specific Sequence ◽

Egfp Gene ◽

Spatio Temporal

ABSTRACTThe CRISPR-Cas9 and related systems offer a unique genome editing tool allowing facile and efficient introduction of heritable and locus-specific sequence modifications in the genome. Despite its molecular precision, temporal and spatial control of gene editing with CRISPR-Cas9 system is very limited. We developed a light-sensitive liposome delivery system that offers a high degree of spatial and temporal control of gene editing with CRISPR/Cas9 system. We demonstrated its high transfection efficiency, by assessing the targeted knockout of eGFP gene in human HEK293 cells (52.8% knockout). We further validated our results at a single-cell resolution using an in vivo eGFP reporter system in zebrafish (77% knockout). To the best of our knowledge we reported the first proof-of-concept of spatio-temporal control of CRISPR/Cas9 by using light-triggered liposomes in both in vitro and in vivo environment.

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Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽

10.3390/biology10060530 ◽

2021 ◽

Vol 10 (6) ◽

pp. 530

Author(s):

Marlo K. Thompson ◽

Robert W. Sobol ◽

Aishwarya Prakash

Keyword(s):

Dna Repair ◽

Mammalian Cells ◽

Genome Stability ◽

Gene Editing ◽

Adaptive Immune System ◽

Zinc Finger Nucleases ◽

Specific Gene ◽

Target Sequence ◽

Transcription Activator ◽

Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

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Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22031391 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1391

Author(s):

Andrey Kropotov ◽

Veronika Kulikova ◽

Kirill Nerinovski ◽

Alexander Yakimov ◽

Maria Svetlova ◽

...

Keyword(s):

Nicotinic Acid ◽

Mammalian Cells ◽

Experimental Models ◽

The Body ◽

Nucleoside Transporters ◽

Hek293 Cells ◽

Nucleoside Transporter ◽

Vitamin B3 ◽

Animal Cells ◽

Nicotinamide Riboside

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

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DNA-binding specificity of GATA family transcription factors.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3999 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3999-4010 ◽

Cited By ~ 448

Author(s):

M Merika ◽

S H Orkin

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Mammalian Cells ◽

Chimeric Protein ◽

Binding Specificity ◽

Mobility Shift ◽

Binding Domains ◽

Dna Binding Specificity ◽

Mobility Shift Assay ◽

Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

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Investigation of Protein Export in Bifidobacterium breve UCC2003

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.6994-7001.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 6994-7001 ◽

Cited By ~ 32

Author(s):

Laura E. MacConaill ◽

Gerald F. Fitzgerald ◽

Douwe van Sinderen

Keyword(s):

Fusion Proteins ◽

Genomic Library ◽

Shuttle Vector ◽

Staphylococcal Nuclease ◽

Cell Fractionation ◽

Reporter System ◽

Bifidobacterium Breve ◽

Positive Effects ◽

Encoding Gene ◽

Export Signal

ABSTRACT The molecular interactions between the bifidobacterial cell and its natural environment, namely, the gastrointestinal tract of its host, are particularly important in understanding the presumed positive effects of Bifidobacterium on the health status of the host. In this study an export-specific reporter system, designed for use in gram-positive organisms and based on the use of the staphylococcal nuclease (Nuc) as a reporter, was employed to identify exported proteins in Bifidobacterium breve UCC2003. A B. breve genomic library of translational fusions to the Nuc-encoding gene devoid of its own export signal was established in the shuttle vector pFUN (I. Poquet, S. D. Ehrlich, and A. Gruss, J. Bacteriol. 180:1904-1912, 1998) and screened for bifidobacterial export signals. Sequence analysis of the fusion proteins obtained that displayed a nuclease-producing phenotype in both Lactococcus lactis and B. breve predicted the presence of a classical signal peptide and/or single or multiple transmembrane domains, thus indicating that some of the export signals in B. breve are comparable to those used in L. lactis. Cell fractionation studies, zymograms, nuclease assays, and Western blotting were employed to confirm the function of the predicted signals and to determine the location and activity of the exported fusion proteins in B. breve and/or L. lactis.

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Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants

Biochemical Journal ◽

10.1042/bj20050694 ◽

2005 ◽

Vol 393 (1) ◽

pp. 245-254 ◽

Cited By ~ 32

Author(s):

Catherine Martel ◽

Paolo Macchi ◽

Luc Furic ◽

Michael A. Kiebler ◽

Luc Desgroseillers

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Mammalian Cells ◽

Rna Binding ◽

Binding Activity ◽

Nuclear Trafficking ◽

Binding Domains ◽

Bipartite Nuclear Localization Signal ◽

Localization Signal

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

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Expression of Hirudin Fusion Proteins in Mammalian Cells

Circulation ◽

10.1161/01.cir.98.24.2744 ◽

1998 ◽

Vol 98 (24) ◽

pp. 2744-2752 ◽

Cited By ~ 26

Author(s):

Kristian Riesbeck ◽

Daxin Chen ◽

Geoffrey Kemball-Cook ◽

John H. McVey ◽

Andrew J. T. George ◽

...

Keyword(s):

Fusion Proteins ◽

Mammalian Cells

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Dephosphorylation of the C-terminal Tyrosyl Residue of the DNA Damage-related Histone H2A.X Is Mediated by the Protein Phosphatase Eyes Absent

Journal of Biological Chemistry ◽

10.1074/jbc.c900032200 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16066-16070 ◽

Cited By ~ 88

Author(s):

Navasona Krishnan ◽

Dae Gwin Jeong ◽

Suk-Kyeong Jung ◽

Seong Eon Ryu ◽

Andrew Xiao ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Mammalian Cells ◽

Protein Tyrosine Phosphatases ◽

Histone H2a ◽

Eyes Absent ◽

Damage Repair ◽

Damage Response

In mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of γH2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response.

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Complex II subunit SDHD is critical for cell growth and metabolism, which can be partially restored with a synthetic ubiquinone analog

BMC Molecular and Cell Biology ◽

10.1186/s12860-021-00370-w ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Aloka B. Bandara ◽

Joshua C. Drake ◽

David A. Brown

Keyword(s):

Mammalian Cells ◽

Krebs Cycle ◽

Atp Synthesis ◽

Hek293 Cells ◽

Knockout Mutant ◽

Complex Ii ◽

Growth Defects ◽

Transport Chain ◽

Mutant Cells

Abstract Background Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. Results Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. Conclusions Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.

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