Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Scarless engineering of the Drosophila genome near any site-specific integration site.

ABSTRACT: We describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This two-step method combines phiC31 integrase-mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.

SUBMITTER: Feng S

PROVIDER: S-EPMC8045726 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Scarless engineering of the Drosophila genome near any site-specific integration site.

Feng Siqian S Lu Shan S Grueber Wesley B WB Mann Richard S RS

Genetics 20210301 3

We describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This two-step method combines phiC31 integrase-mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this meth ...[more]

PMID: 33772309

Similar Datasets

High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration.

Project description:Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase-assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker-free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.

| S-EPMC10005180 | biostudies-literature

Long-range targeted manipulation of the Drosophila genome by site-specific integration and recombinational resolution.

Project description:Significant advances in genomics underscore the importance of targeted mutagenesis for gene function analysis. Here we have developed a scheme for long-range targeted manipulation of genes in the Drosophila genome. Utilizing an attP attachment site for the phiC31 integrase previously targeted to the nbs gene, we integrated an 80-kb genomic fragment at its endogenous locus to generate a tandem duplication of the region. We achieved reduction to a single copy by inducing recombination via a site-specific DNA break. We report that, despite the large size of the DNA fragment, both plasmid integration and duplication reduction can be accomplished efficiently. Importantly, the integrating genomic fragment can serve as a venue for introducing targeted modifications to the entire region. We successfully introduced a new attachment site 70 kb from the existing attP using this two-step scheme, making a new region susceptible to targeted mutagenesis. By experimenting with different placements of the future DNA break site in the integrating vector, we established a vector configuration that facilitates the recovery of desired modifications. We also show that reduction events can occur efficiently through unequal meiotic crossing over between the large duplications. Based on our results, we suggest that a collection of 1200 lines with attachment sites inserted every 140 kb throughout the genome would render all Drosophila genes amenable to targeted mutagenesis. Excitingly, all of the components involved are likely functional in other eukaryotes, making our scheme for long-range targeted manipulation readily applicable to other systems.

| S-EPMC3567732 | biostudies-literature

Site-specific recombinase genome engineering toolkit in maize.

Project description:Site-specific recombinase enzymes function in heterologous cellular environments to initiate strand-switching reactions between unique DNA sequences termed recombinase binding sites. Depending on binding site position and orientation, reactions result in integrations, excisions, or inversions of targeted DNA sequences in a precise and predictable manner. Here, we established five different stable recombinase expression lines in maize through Agrobacterium-mediated transformation of T-DNA molecules that contain coding sequences for Cre, R, FLPe, phiC31 Integrase, and phiC31 excisionase. Through the bombardment of recombinase activated DsRed transient expression constructs, we have determined that all five recombinases are functional in maize plants. These recombinase expression lines could be utilized for a variety of genetic engineering applications, including selectable marker removal, targeted transgene integration into predetermined locations, and gene stacking.

| S-EPMC7061458 | biostudies-literature

Genome engineering in Bacillus anthracis using tyrosine site-specific recombinases.

Project description:Tyrosine site-specific recombinases (T-SSR) are polynucleotidyltransferases that catalyze cutting and joining reactions between short specific DNA sequences. We developed three systems for performing genetic modifications in Bacillus anthracis that use T-SSR and their cognate target sequences, namely Escherichia coli bacteriophage P1 Cre-loxP, Saccharomyces cerevisiae Flp-FRT, and a newly discovered IntXO-PSL system from B. anthracis plasmid pXO1. All three tyrosine recombinase systems were used for creation of a B. anthracis sporulation-deficient, plasmid-free strain deleted for ten proteases which had been identified by proteomic analysis as being present in the B. anthracis secretome. This strain was used successfully for production of various recombinant proteins, including several that are candidates for inclusion in improved anthrax vaccines. These genetic tools developed for DNA manipulation in B. anthracis were also used for construction of strains having chromosomal insertions of 1, 2, or 3 adjacent atxA genes. AtxA is a B. anthracis global transcriptional regulator required for the response of B. anthracis virulence factor genes to bicarbonate. We found a positive correlation between the atxA copy number and the expression level of the pagA gene encoding B. anthracis protective antigen, when strains were grown in a carbon dioxide atmosphere. These results demonstrate that the three T-SSR systems described here provide effective tools for B. anthracis genome editing. These T-SSR systems may also be applicable to other prokaryotes and to eukaryotes.

| S-EPMC5567495 | biostudies-literature

Site-specific Integration Ushers in a New Era of Precise CHO Cell Line Engineering.

Project description:Chinese hamster ovary (CHO) cells are widely used for the production of therapeutic proteins. Customarily, CHO production cell lines are established through random integration, which requires laborious screening of many clones to isolate suitable producers. In contrast, site-specific integration (SSI) accelerates cell line development by targeting integration of transgenes to pre-validated genomic loci capable of supporting high and stable expression. To date, a relatively small number of these so called 'hot spots' have been identified, mainly through empirical methods. Nevertheless, nuclease-mediated and recombinase-mediated SSI have revolutionized cell line engineering by enabling rational and reproducible transgene targeting.

| S-EPMC6510493 | biostudies-literature

Understanding functional miRNA-target interactions in vivo by site-specific genome engineering.

Project description:MicroRNA (miRNA) target recognition is largely dictated by short 'seed' sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA-target interactions to the regulation of biological processes in vivo remains challenging. Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA-MRE interactions at any point during development.

| S-EPMC4143950 | biostudies-literature

VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering.

Project description:We developed two new site-specific recombination systems named VCre/VloxP and SCre/SloxP for genome engineering. Their recognition sites are different from Cre recognition sites because VCre and SCre recombinases share less protein similarity with Cre, even though the basic 13-8-13 structures of their recognition sites are identical. Mutant VloxP and SloxP, which have the same uses as mutant loxP, were also developed. VCre/VloxP and SCre/SloxP in combination with Cre/loxP and Flp/FRT systems can serve as powerful tools for genome engineering, especially when used to genetically modify both alleles of a single gene in mouse and human cells.

| S-EPMC3082901 | biostudies-literature

Multiplexed site-specific genome engineering in <i>Mycolicibacterium neoaurum</i> by Att/Int system.

Project description:Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories. For some uncommon microbial hosts, such as Mycolicibacterium and Mycobacterium species, however, it is a challenge. Here, we present a multiplexed integrase-assisted site-specific recombination (miSSR) method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories. First, a single-step multi-copy integration method was established in M. neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy, the efficiencies of which were ∼100% for no more than three-copy integration events and decreased sharply to ∼20% for five-copy integration events. Second, the R4, Bxb1 and ΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events. Third, a reconstructed mycolicibacterial Xer recombinases (Xer-cise) system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation. As a proof of concept, the biosynthetic pathway of ergothioneine (EGT) in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system. With six copies of the biosynthetic gene clusters (BGCs) of EGT and pentose phosphate isomerase (PRT), the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L, which was 3.77 times of that in the wild strain. The improvements indicated that the miSSR system was an effective, flexible, and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.

| S-EPMC9213222 | biostudies-literature

An efficient, scarless, selection-free technology for phage engineering.

Project description:Most recently developed phage engineering technologies are based on the CRISPR-Cas system. Here, we present a non-CRISPR-based method for genetically engineering the Escherichia coli phages T5, T7, P1, and λ by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes. The technology comprises E. coli harbouring a plasmid encoding a potent recombinase and a gene transiently silencing a repair system. Oligonucleotides with the desired phage mutation are electroporated into E. coli followed by infection of the target bacteriophage. The high efficiency of this technology, which yields 1-14% of desired recombinants, allows low-throughput screening for the desired mutant. We have demonstrated the use of this technology for single-base substitutions, for deletions of 50-201 bases, for insertions of 20 bases, and for four different phages. The technology may also be readily modified for use across many additional bacterial and phage strains.[Figure: see text].

| S-EPMC10583621 | biostudies-literature

Site-specific genomic targeting in Drosophila.

Project description:Transcriptional regulation of transgenes depends upon genomic localization in higher eukaryotes. For the applied use of transgenic organisms as producers of pharmaceutically relevant proteins or as pest population control agents, a method to make transgene expression predictable is highly desirable. A targeting method that allows precise cassette replacement comprising solely genes of interest (without extraneous donor vector sequences) would be highly advantageous for insects and other multicellular organisms. In this report, we describe a method for transgene targeting to predefined chromosomal sites in Drosophila by using a transposon vector that, once integrated in the germ line, acts as an acceptor site for donor vectors. To make recombinational insertions irreversible, a FLP recombinase-mediated cassette exchange strategy was used, and to enhance donor-target pairing, a homing sequence from the linotte locus was used. Site-specific recombinants were screened by interconvertible eye fluorescence marker phenotypes yielding, on average, targeted insertions at a frequency of 23%. The cassette exchange system provides for repetitive integrations into the same locus, allowing comparative analysis of true transgenic alleles. Furthermore, this method was used to stabilize a targeted transgene by the postintegration excision of putatively mobile transposon sequences. The genomic targeting and stabilization strategy described for Drosophila should be applicable to other insects, specifically for the goals of optimizing heterologous protein expression and enhancing ecological safety of transgenic strains intended for release in biocontrol programs.

| S-EPMC1194931 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data