scholarly journals Fast and cloning-free CRISPR/Cas9-mediated genomic editing in mammalian cells

2019 ◽  
Author(s):  
Paul T. Manna ◽  
Luther J. Davis ◽  
Margaret S. Robinson
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
End Joining ◽  
Genomic Locus ◽  
Product Integration ◽  
Strand Breaks ◽  
Guide Rna ◽  
Pcr Product ◽  
Hands On ◽  
Genomic Editing

SummaryCHoP-In (CRISPR/Cas9-mediated, Homology-independent, PCR-product Integration) is a fast and cloning-free strategy for genomic editing of mammalian cells. The desired integration fragment is produced as a PCR product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9/guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of key membrane trafficking proteins. The lack of any donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.

scholarly journals Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks

2020 ◽  
Vol 21 (18) ◽  
pp. 6461
Author(s):  
Han Yang ◽  
Shuling Ren ◽  
Siyuan Yu ◽  
Haifeng Pan ◽  
Tingdong Li ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Initial Step ◽  
Double Strand Breaks ◽  
Repair Pathway ◽  
End Joining ◽  
Strand Breaks ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Repair Pathways

Precise gene editing is—or will soon be—in clinical use for several diseases, and more applications are under development. The programmable nuclease Cas9, directed by a single-guide RNA (sgRNA), can introduce double-strand breaks (DSBs) in target sites of genomic DNA, which constitutes the initial step of gene editing using this novel technology. In mammals, two pathways dominate the repair of the DSBs—nonhomologous end joining (NHEJ) and homology-directed repair (HDR)—and the outcome of gene editing mainly depends on the choice between these two repair pathways. Although HDR is attractive for its high fidelity, the choice of repair pathway is biased in a biological context. Mammalian cells preferentially employ NHEJ over HDR through several mechanisms: NHEJ is active throughout the cell cycle, whereas HDR is restricted to S/G2 phases; NHEJ is faster than HDR; and NHEJ suppresses the HDR process. This suggests that definitive control of outcome of the programmed DNA lesioning could be achieved through manipulating the choice of cellular repair pathway. In this review, we summarize the DSB repair pathways, the mechanisms involved in choice selection based on DNA resection, and make progress in the research investigating strategies that favor Cas9-mediated HDR based on the manipulation of repair pathway choice to increase the frequency of HDR in mammalian cells. The remaining problems in improving HDR efficiency are also discussed. This review should facilitate the development of CRISPR/Cas9 technology to achieve more precise gene editing.

scholarly journals Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
Akira Sassa ◽  
Lalith Perera ◽  
David D. Shock ◽  
William A. Beard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Strand Break ◽  
Structural Basis ◽  
End Joining ◽  
Strand Breaks ◽  
Nucleotide Pools ◽  
Nucleotide Insertion ◽  
Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

DNA double-strand break repair within heterochromatic regions

2012 ◽  
Vol 40 (1) ◽  
pp. 173-178 ◽  
Author(s):  
Johanne M. Murray ◽  
Tom Stiff ◽  
Penny A. Jeggo
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
Strand Break ◽  
Higher Order ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Dna Dsbs ◽  
A Cell ◽  
Non Homologous End Joining

DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.

scholarly journals Genome-wide Cas9 binding specificity in Saccharomyces cerevisiae

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9442 ◽  
Author(s):  
Zachary J. Waldrip ◽  
Piroon Jenjaroenpun ◽  
Oktawia DeYoung ◽  
Intawat Nookaew ◽  
Sean D. Taverna ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Specificity ◽  
Genomic Locus ◽  
Site Specific ◽  
Guide Rna ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Chromosome Iii ◽  
Genomic Editing

The CRISPR system has become heavily utilized in biomedical research as a tool for genomic editing as well as for site-specific chromosomal localization of specific proteins. For example, we developed a CRISPR-based methodology for enriching a specific genomic locus of interest for proteomic analysis in Saccharomyces cerevisiae, which utilized a guide RNA-targeted, catalytically dead Cas9 (dCas9) as an affinity reagent. To more comprehensively evaluate the genomic specificity of using dCas9 as a site-specific tool for chromosomal studies, we performed dCas9-mediated locus enrichment followed by next-generation sequencing on a genome-wide scale. As a test locus, we used the ARS305 origin of replication on chromosome III in S. cerevisiae. We found that enrichment of this site is highly specific, with virtually no off-target enrichment of unique genomic sequences. The high specificity of genomic localization and enrichment suggests that dCas9-mediated technologies have promising potential for site-specific chromosomal studies in organisms with relatively small genomes such as yeasts.

Regulation of the cellular DNA double-strand break response

2007 ◽  
Vol 85 (6) ◽  
pp. 663-674 ◽  
Author(s):  
Kendra L. Cann ◽  
Geoffrey G. Hicks
Keyword(s):  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Cell Cycle Checkpoints ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Cellular Dna

DNA double-strand breaks occur frequently in cycling cells, and are also induced by exogenous sources, including ionizing radiation. Cells have developed integrated double-strand break response pathways to cope with these lesions, including pathways that initiate DNA repair (either via homologous recombination or nonhomologous end joining), the cell-cycle checkpoints (G1–S, intra-S phase, and G2–M) that provide time for repair, and apoptosis. However, before any of these pathways can be activated, the damage must first be recognized. In this review, we will discuss how the response of mammalian cells to DNA double-strand breaks is regulated, beginning with the activation of ATM, the pinnacle kinase of the double-strand break signalling cascade.

scholarly journals DNA Substrate Dependence of p53-Mediated Regulation of Double-Strand Break Repair

2002 ◽  
Vol 22 (17) ◽  
pp. 6306-6317 ◽  
Author(s):  
Nuray Akyüz ◽  
Gisa S. Boehden ◽  
Silke Süsse ◽  
Andreas Rimek ◽  
Ute Preuss ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
P53 Expression ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Multistep Process ◽  
Non Homologous End Joining

ABSTRACT DNA double-strand breaks (DSBs) arise spontaneously after the conversion of DNA adducts or single-strand breaks by DNA repair or replication and can be introduced experimentally by expression of specific endonucleases. Correct repair of DSBs is central to the maintenance of genomic integrity in mammalian cells, since errors give rise to translocations, deletions, duplications, and expansions, which accelerate the multistep process of tumor progression. For p53 direct regulatory roles in homologous recombination (HR) and in non-homologous end joining (NHEJ) were postulated. To systematically analyze the involvement of p53 in DSB repair, we generated a fluorescence-based assay system with a series of episomal and chromosomally integrated substrates for I-SceI meganuclease-triggered repair. Our data indicate that human wild-type p53, produced either stably or transiently in a p53-negative background, inhibits HR between substrates for conservative HR (cHR) and for gene deletions. NHEJ via microhomologies flanking the I-SceI cleavage site was also downregulated after p53 expression. Interestingly, the p53-dependent downregulation of homology-directed repair was maximal during cHR between sequences with short homologies. Inhibition was minimal during recombination between substrates that support reporter gene reconstitution by HR and NHEJ. p53 with a hotspot mutation at codon 281, 273, 248, 175, or 143 was severely defective in regulating DSB repair (frequencies elevated up to 26-fold). For the transcriptional transactivation-inactive variant p53(138V) a defect became apparent with short homologies only. These results suggest that p53 plays a role in restraining DNA exchange between imperfectly homologous sequences and thereby in suppressing tumorigenic genome rearrangements.

Double-Strand Breaks Repair by Non-Homologous DNA End Joining in Mammalian Cells

2006 ◽  
Vol 7 (5) ◽  
pp. 311-322 ◽  
Author(s):  
Mariusz Malinowski ◽  
Elzbieta Pastwa
Keyword(s):  
Mammalian Cells ◽  
Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks

scholarly journals Microhomology-mediated CRISPR/Cas9-based method for genome editing in fission yeast

2018 ◽  
Author(s):  
Aki Hayashi ◽  
Katsunori Tanaka
Keyword(s):  
Fission Yeast ◽  
Genome Editing ◽  
High Frequency ◽  
Target Genes ◽  
Point Mutations ◽  
Inducible Promoter ◽  
Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Guide Rna

The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the microhomology mediated end joining (MMEJ)-based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the MMEJ-mediated genome editing and found that the MMEJ-mediated method provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing microhomology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.

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