Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision

Caenorhabditis eleganshas a complete annotated genome sequence that is augmented by increasing quantities of data from high-throughput postgenomic analyses. This has led to an increasing need to identify the biological functions of specific genes using reverse genetics, i.e., moving from gene to phenotype. Fundamental to this aim is the ability to alter the structure of particular genes by means that are not accessible to classical genetic strategies. Thus, one dream ofC. elegansresearchers is to establish a toolkit for the controlled manipulation of anylociwithin the genome. AlthoughC. elegansis amenable to a wide variety of genetic and molecular manipulations, controlled manipulation of endogenous genes by, for example, gene targeting has proved elusive until relatively recently. In this review, we describe and discuss the different methods available for the inactivation and modification of endogenous loci with a focus on strategies that permit some measure of control in this process. We describe methods that use random mutagenesis to isolate mutations in specific genes. We then focus on techniques that allow controlled manipulation of the genome: gene modification by transposon mobilisation, gene knock-out mediated by zinc-finger nucleases, and gene targeting by biolistic transformation.

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Faculty Opinions recommendation of Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.719711737.793500652 ◽

2014 ◽

Author(s):

Renate Schmidt

Keyword(s):

Genome Editing ◽

Gene Targeting ◽

Marker Excision

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A simplified counter-selection recombineering protocol for creating fluorescent protein reporter constructs directly from C. elegans fosmid genomic clones

BMC Biotechnology ◽

10.1186/1472-6750-13-1 ◽

2013 ◽

Vol 13 (1) ◽

pp. 1 ◽

Cited By ~ 19

Author(s):

Nisha Hirani ◽

Marcel Westenberg ◽

Minaxi S Gami ◽

Paul Davis ◽

Ian A Hope ◽

...

Keyword(s):

Fluorescent Protein ◽

Genomic Clones ◽

C Elegans ◽

Counter Selection ◽

Fluorescent Protein Reporter

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Use of a Sibling Subtraction Method (SSM) for Identifying Causal Mutations in C. elegans by Whole-Genome Sequencing

10.1101/166355 ◽

2017 ◽

Author(s):

Braveen B. Joseph ◽

Nicolas A. Blouin ◽

David S. Fay

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Synthetic Lethality ◽

Dna Lesions ◽

Screening Methods ◽

Whole Genome ◽

Subtraction Method ◽

C Elegans ◽

Density Mapping ◽

Counter Selection

AbstractWhole-genome sequencing (WGS) has become an indispensible tool for identifying causal mutations obtained from genetic screens. To reduce the number of causal mutation candidates typically uncovered by WGS, C. elegans researchers have developed several strategies. One involves crossing N2-background mutants to the polymorphic strain CB4856, which can be used to simultaneously identify mutant-strain variants and obtain high-density mapping information. This approach, however, is not well suited for uncovering mutations in complex genetic backgrounds, and CB4856 polymorphisms can alter phenotypes. Several other approaches make use of DNA variants introduced by mutagenesis. This information is used to implicate genomic regions with high densities of DNA lesions that persist after mutant backcrossing, but these methods provide lower mapping resolution than use of CB4856. To identify suppressor mutations using WGS, we developed a new approach termed the Sibling Subtraction Method (SSM). This method works by eliminating variants present in both mutants and their non-mutant siblings, thus greatly reducing the number of candidates. We used this method here with two members of the C. elegans NimA-related kinase family, nekl-2 and nekl-3. Combining weak aphenotypic alleles of nekl-2 and nekl-3 leads to penetrant molting defects and larval arrest. We isolated ~50 suppressors of nekl-2; nekl-3 synthetic lethality using F1-clonal screening methods and a toxin-based counter-selection strategy. When applied to five of the identified suppressors, SSM led to only one to four suppressor candidates per strain. Thus SSM is a powerful approach for identifying causal mutations in any genetic background and provides an alternative to current methods.

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Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants

Frontiers in Genome Editing ◽

10.3389/fgeed.2020.617713 ◽

2021 ◽

Vol 2 ◽

Author(s):

Namie Ohtsuki ◽

Keiko Kizawa ◽

Akiko Mori ◽

Ayako Nishizawa-Yokoi ◽

Takao Komatsuda ◽

...

Keyword(s):

Genome Editing ◽

Gene Targeting ◽

Mirna Target ◽

Single Strand ◽

Target Site ◽

Selection Marker ◽

Base Substitution ◽

Marker Excision ◽

Single Strand Annealing ◽

Strand Annealing

Gene targeting (GT) enables precise genome modification—e.g., the introduction of base substitutions—using donor DNA as a template. Combined with clean excision of the selection marker used to select GT cells, GT is expected to become a standard, generally applicable, base editing system. Previously, we demonstrated marker excision via a piggyBac transposon from GT-modified loci in rice. However, piggyBac-mediated marker excision has the limitation that it recognizes only the sequence TTAA. Recently, we proposed a novel and universal precise genome editing system consisting of GT with subsequent single-strand annealing (SSA)-mediated marker excision, which has, in principle, no limitation of target sequences. In this study, we introduced base substitutions into the microRNA miR172 target site of the OsCly1 gene—an ortholog of the barley Cleistogamy1 gene involved in cleistogamous flowering. To ensure efficient SSA, the GT vector harbors 1.2-kb overlapped sequences at both ends of a selection marker. The frequency of positive–negative selection-mediated GT using the vector with overlapped sequences was comparable with that achieved using vectors for piggyBac-mediated marker excision without overlapped sequences, with the frequency of SSA-mediated marker excision calculated as ~40% in the T0 generation. This frequency is thought to be adequate to produce marker-free cells, although it is lower than that achieved with piggyBac-mediated marker excision, which approaches 100%. To date, introduction of precise substitutions in discontinuous multiple bases of a targeted gene using base editors and the prime editing system based on CRISPR/Cas9 has been quite difficult. Here, using GT and our SSA-mediated marker excision system, we succeeded in the precise base substitution not only of single bases but also of artificial discontinuous multiple bases in the miR172 target site of the OsCly1 gene. Precise base substitution of miRNA target sites in target genes using this precise genome editing system will be a powerful tool in the production of valuable crops with improved traits.

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Deletion and Allelic Exchange of the Aspergillus fumigatus veA Locus via a Novel Recyclable Marker Module

Eukaryotic Cell ◽

10.1128/ec.4.7.1298-1307.2005 ◽

2005 ◽

Vol 4 (7) ◽

pp. 1298-1307 ◽

Cited By ~ 89

Author(s):

Sven Krappmann ◽

Özgür Bayram ◽

Gerhard H. Braus

Keyword(s):

Aspergillus Fumigatus ◽

Opportunistic Pathogen ◽

Dependent Manner ◽

Wild Type ◽

Gene Deletions ◽

Counter Selection ◽

Marker Excision ◽

Detailed Evaluation ◽

Selection Of ◽

Selection Of Transformants

ABSTRACT Detailed evaluation of gene functions in an asexual fungus requires advanced methods of molecular biology. For the generation of targeted gene deletions in the opportunistic pathogen Aspergillus fumigatus we designed a novel blaster module allowing dominant selection of transformants due to resistance to phleomycin as well as dominant (counter)selection of a Cre recombinase-mediated marker excision event. For validation purposes we have deleted the A. fumigatus pabaA gene in a wild-type isolate by making use of this cassette. The resulting pabaA::loxP strain served as the recipient for subsequent targeting of the velvet locus. Homologous reconstitution of the deleted gene was performed by an allele whose expression is driven in a nitrogen source-dependent manner, as validated by Northern analyses. Overexpression of the veA locus in A. fumigatus does not result in any obvious phenotype, whereas the sporulation capacities of the veA null mutant are reduced on nitrate-containing medium, a phenotype that is completely restored in the reconstituted strain.

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The glycomes of Caenorhabditis elegans and other model organisms

Biochemical Society Symposium ◽

10.1042/bss0690117 ◽

2002 ◽

Vol 69 ◽

pp. 117-134 ◽

Cited By ~ 47

Author(s):

Stuart M. Haslam ◽

David Gems ◽

Howard R. Morris ◽

Anne Dell

Keyword(s):

Caenorhabditis Elegans ◽

Current Knowledge ◽

Structural Data ◽

Model Organisms ◽

Entire Genome ◽

C Elegans ◽

The Face ◽

Area Of Concern ◽

Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

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How do histone modifications contribute to transgenerational epigenetic inheritance in C. elegans?

Biochemical Society Transactions ◽

10.1042/bst20190944 ◽

2020 ◽

Vol 48 (3) ◽

pp. 1019-1034 ◽

Cited By ~ 1

Author(s):

Rachel M. Woodhouse ◽

Alyson Ashe

Keyword(s):

Histone Modifications ◽

Molecular Mechanisms ◽

Epigenetic Inheritance ◽

Nematode Caenorhabditis Elegans ◽

Histone Methyltransferases ◽

Transgenerational Epigenetic Inheritance ◽

C Elegans ◽

Recent Developments ◽

Gene Regulatory

Gene regulatory information can be inherited between generations in a phenomenon termed transgenerational epigenetic inheritance (TEI). While examples of TEI in many animals accumulate, the nematode Caenorhabditis elegans has proven particularly useful in investigating the underlying molecular mechanisms of this phenomenon. In C. elegans and other animals, the modification of histone proteins has emerged as a potential carrier and effector of transgenerational epigenetic information. In this review, we explore the contribution of histone modifications to TEI in C. elegans. We describe the role of repressive histone marks, histone methyltransferases, and associated chromatin factors in heritable gene silencing, and discuss recent developments and unanswered questions in how these factors integrate with other known TEI mechanisms. We also review the transgenerational effects of the manipulation of histone modifications on germline health and longevity.

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Centrosome Aurora A gradient ensures single polarity axis in C. elegans embryos

Biochemical Society Transactions ◽

10.1042/bst20200298 ◽

2020 ◽

Vol 48 (3) ◽

pp. 1243-1253 ◽

Cited By ~ 1

Author(s):

Sukriti Kapoor ◽

Sachin Kotak

Keyword(s):

Symmetry Breaking ◽

Cell Fate ◽

Cell Cortex ◽

Axis Formation ◽

Aurora A Kinase ◽

Aurora A ◽

C Elegans ◽

Cell Embryo ◽

Polarity Establishment

Cellular asymmetries are vital for generating cell fate diversity during development and in stem cells. In the newly fertilized Caenorhabditis elegans embryo, centrosomes are responsible for polarity establishment, i.e. anterior–posterior body axis formation. The signal for polarity originates from the centrosomes and is transmitted to the cell cortex, where it disassembles the actomyosin network. This event leads to symmetry breaking and the establishment of distinct domains of evolutionarily conserved PAR proteins. However, the identity of an essential component that localizes to the centrosomes and promotes symmetry breaking was unknown. Recent work has uncovered that the loss of Aurora A kinase (AIR-1 in C. elegans and hereafter referred to as Aurora A) in the one-cell embryo disrupts stereotypical actomyosin-based cortical flows that occur at the time of polarity establishment. This misregulation of actomyosin flow dynamics results in the occurrence of two polarity axes. Notably, the role of Aurora A in ensuring a single polarity axis is independent of its well-established function in centrosome maturation. The mechanism by which Aurora A directs symmetry breaking is likely through direct regulation of Rho-dependent contractility. In this mini-review, we will discuss the unconventional role of Aurora A kinase in polarity establishment in C. elegans embryos and propose a refined model of centrosome-dependent symmetry breaking.

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