Dataset Information

Genome targeting by hybrid Flp-TAL recombinases.

ABSTRACT: Genome engineering is a rapidly evolving field that benefits from the availability of different tools that can be used to perform genome manipulation tasks. We describe here the development of the Flp-TAL recombinases that can target genomic FRT-like sequences in their native chromosomal locations. Flp-TAL recombinases are hybrid enzymes that are composed of two functional modules: a variant of site-specific tyrosine recombinase Flp, which can have either narrow or broad target specificity, and the DNA-binding domain of the transcription activator-like effector, TAL. In Flp-TAL, the TAL module is responsible for delivering and stabilizing the Flp module onto the desired genomic FRT-like sequence where the Flp module mediates recombination. We demonstrate the functionality of the Flp-TAL recombinases by performing integration and deletion experiments in human HEK-293 cells. In the integration experiments we targeted a vector to three genomic FRT-like sequences located in the ?-globin locus. In the deletion experiments we excised ~?15 kilobases of DNA that contained a fragment of the integrated vector sequence and the neighboring genome sequence. On average, the efficiency of the integration and deletion reactions was about 0.1% and 20%, respectively.

SUBMITTER: Voziyanova E

PROVIDER: S-EPMC7562724 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Genome targeting by hybrid Flp-TAL recombinases.

Voziyanova Eugenia E Li Feng F Shah Riddhi R Voziyanov Yuri Y

Scientific reports 20201015 1

Genome engineering is a rapidly evolving field that benefits from the availability of different tools that can be used to perform genome manipulation tasks. We describe here the development of the Flp-TAL recombinases that can target genomic FRT-like sequences in their native chromosomal locations. Flp-TAL recombinases are hybrid enzymes that are composed of two functional modules: a variant of site-specific tyrosine recombinase Flp, which can have either narrow or broad target specificity, and ...[more]

PMID: 33060660

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Project description:FMRFamide-like peptide (FLP) signalling systems are core to nematode neuromuscular function. Novel drug discovery efforts associated with nematode FLP/FLP receptor biology are advanced through the accumulation of basic biological data that can reveal subtle complexities within the neuropeptidergic system. This study reports the characterisation of FMRFamide-like peptide encoding gene-11 (flp-11) and FMRFamide-like peptide encoding gene-32 (flp-32), two distinct flp genes which encode the analogous peptide, AMRN(A/S)LVRFamide, in multiple nematode species - the only known example of this phenomenon within the FLPergic system of nematodes. Using bioinformatics, in situ hybridisation, immunocytochemistry and behavioural assays we show that: (i) flp-11 and -32 are distinct flp genes expressed individually or in tandem across multiple nematode species, where they encode a highly similar peptide; (ii) flp-11 does not appear to be the most widely expressed flp in Caenorhabditis elegans; (iii) in species expressing both flp-11 and flp-32, flp-11 displays a conserved, restricted expression pattern across nematode clades and lifestyles; (iv) in species expressing both flp-11 and flp-32, flp-32 expression is more widespread and less conserved than flp-11; (v) in species expressing only flp-11, the flp-11 expression profile is more similar to the flp-32 profile observed in species expressing both; and (vi) FLP-11 peptides inhibit motor function in multiple nematode species. The biological significance and evolutionary origin of flp-11 and -32 peptide duplication remains unclear despite attempts to identify a common ancestor; this may become clearer as the availability of genomic data improves. This work provides insight into the complexity of the neuropeptidergic system in nematodes, and begins to examine how nematodes may compensate for structural neuronal simplicity. From a parasite control standpoint, this work underscores the importance of basic biological data, and has wider implications for the utility of C. elegans as a model for parasite neurobiology.

Dataset Information

Genome targeting by hybrid Flp-TAL recombinases.

Publications

Genome targeting by hybrid Flp-TAL recombinases.

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