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Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking

ABSTRACT: Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking

PROVIDER: PRJNA557250 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10359715_1.fastq.gz	Fastqsanger.gz
	SRR10359715_2.fastq.gz	Fastqsanger.gz
	SRR10359716_1.fastq.gz	Fastqsanger.gz
	SRR10359716_2.fastq.gz	Fastqsanger.gz
	SRR10359717_1.fastq.gz	Fastqsanger.gz

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Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking

Project description:Genome editing typically involves recombination between donor nucleic acids and genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, amongst other deleterious by-products, DSBs yield translocations, off-target mutations and, most pervasively, unpredictable on-target allelic disruptions. Remarkably, hitherto, the untoward phenotypic consequences of on-target disruptions at allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness due to on-target DSBs and report that simultaneous single-stranded DNA break formation at donor and target DNA by CRISPR-Cas9 “nickases” overcomes, to a great extent, such genotype-phenotype disrupting events. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments (i.e. live-cell reporter tags) into essential and multiple-copy genomic sequences while circumventing most of the allelic and non-allelic collateral mutations and chromosomal rearrangements characteristic of nuclease-dependent gene editing procedures.

2019-12-04 | GSE135064 | GEO

Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking.

Project description:Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR-Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein by genotyping assays and high-throughput genome-wide sequencing of DNA translocations, this is achieved while circumventing most allelic and non-allelic mutations and chromosomal rearrangements characteristic of nuclease-dependent procedures. Our work demonstrates that in trans paired nicking retains target protein dosages in gene-edited cell populations and expands gene editing to chromosomal tracts previously not possible to modify seamlessly due to their recurrence in the genome or essentiality for cell function.

| S-EPMC6954423 | biostudies-literature

In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting.

Project description:Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.CRISPR-Cas9-based gene editing involves double-strand breaks at target sequences, which are often repaired by mutagenic non-homologous end-joining. Here the authors use Cas9 nickases to generate coordinated single-strand breaks in donor and target DNA for precise homology-directed gene editing.

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Gene expression changes in cutting and inverted cutting in Populus yunnanensis at the primary and rapid growth stages respectively

Project description:Illumina technology was used to generate mRNA profiles of stem apex of Populus yunnanensis with cutting and inverted cutting. Total RNA was extracted separately from each plant and pooled to three biological replicates per condition. RNA concentration and purity was measured using NanoDrop 2000(Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

2023-12-08 | GSE249347 | GEO

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Project description:Cis-trans

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Caste-specific RNA editomes in the leaf-cutting ant Acromyrmex echinatior

Project description:Eusocial insects have evolved the capacity to generate adults with distinct morphological, reproductive and behavioural phenotypes from the same genome. Recent studies suggest that RNA editing might enhance the diversity of gene products at the post-transcriptional level, particularly to induce functional changes in the nervous system. Using head samples from the leaf-cutting ant Acromyrmex echinatior, we compare RNA editomes across eusocial castes, identifying ca. 11,000 RNA editing sites in gynes, large workers and small workers. Those editing sites map to 800 genes functionally enriched for neurotransmission, circadian rhythm, temperature response, RNA splicing and carboxylic acid biosynthesis. Most A. echinatior editing sites are species specific, but 8M-bM-^@M-^S23% are conserved across ant subfamilies and likely to have been important for the evolution of eusociality in ants. The level of editing varies for the same site between castes, suggesting that RNA editing might be a general mechanism that shapes caste behaviour in ants. Analysis of genome-wide RNA editing in three different female castes of the the leaf-cutting ant Acromyrmex echinatior.

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Differential Expression of miRNAs in Bovine Skeletal Muscle that Display Varying Degrees of the Dark-cutting Phenotype.

Project description:Identify specific genetic regulatory and signaling mechanisms that result in occurrence of “dark cutting meat” from beef carcasses in order to develop novel strategies for reducing incidence

2022-01-07 | GSE193003 | GEO

Paired CRISPR screens to map gene regulation in cis and trans (HiChIP)

Project description:In this study, we develop a scalable approach that pairs discovery of cis- and trans-acting elements to systematically decipher the gene regulatory network. We explore the regulation of the immune checkpoint PD-L1 under both basal and IFNγ-stimulated contexts. PD-L1 expression on tumor cells serves as a clinically-actionable diagnostic for immune checkpoint blockade therapy across multiple cancer types.

2024-11-28 | GSE255700 | GEO

Expanding the cultivated human archaeome

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Expanding the molecular versatility of an optogenetic switch in yeast.

Project description:In the budding yeast Saccharomyces cerevisiae, the FUN-LOV (FUNgal Light Oxygen and Voltage) optogenetic switch enables high levels of light-activated gene expression in a reversible and tunable fashion. The FUN-LOV components, under identical promoter and terminator sequences, are encoded in two different plasmids, which limits its future applications in wild and industrial yeast strains. In this work, we aim to expand the molecular versatility of the FUN-LOV switch to increase its biotechnological applications. Initially, we generated new variants of this system by replacing the promoter and terminator sequences and by cloning the system in a single plasmid (FUN-LOVSP). In a second step, we included the nourseothricin (Nat) or hygromycin (Hph) antibiotic resistances genes in the new FUN-LOVSP plasmid, generating two new variants (FUN-LOVSP-Nat and FUN-LOVSP-Hph), to allow selection after genome integration. Then, we compared the levels of light-activated expression for each FUN-LOV variants using the luciferase reporter gene in the BY4741 yeast strain. The results indicate that FUN-LOVSP-Nat and FUN-LOVSP-Hph, either episomally or genome integrated, reached higher levels of luciferase expression upon blue-light stimulation compared the original FUN-LOV system. Finally, we demonstrated the functionality of FUN-LOVSP-Hph in the 59A-EC1118 wine yeast strain, showing similar levels of reporter gene induction under blue-light respect to the laboratory strain, and with lower luciferase expression background in darkness condition. Altogether, the new FUN-LOV variants described here are functional in different yeast strains, expanding the biotechnological applications of this optogenetic tool.

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