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Engineering spacer specificity of the Cre/loxP system

ABSTRACT: Translational research on the Cre/loxP recombination system focuses on enhancing its specificity by modifying Cre/DNA interactions. Despite extensive efforts, the exact mechanisms governing how Cre distinguishes between substrates remains elusive. Cre recognizes 13 bp inverted repeats, initiating recombination in the 8 bp spacer region. While literature suggests that efficient recombination proceeds between lox sites with non-loxP spacer sequences when both lox sites have matching spacers, experimental validation for this assumption is lacking. To fill this gap, we investigated target site variations of identical pairs of the loxP 8 bp spacer region, screening 6,000 unique loxP-like sequences. Approximately 84% of these sites exhibited efficient recombination, affirming the flexibility of spacer sequences for catalysis. However, certain spacers negatively impacted recombination, emphasizing sequence dependence. Directed evolution of Cre on inefficiently recombined spacers not only yielded recombinases with enhanced activity but also mutants with reprogrammed selective activity. Mutations altering spacer specificity were identified, and molecular modelling and dynamics simulations elucidated the mechanism behind this specificity switch. These findings highlight the potential to fine-tune site-specific recombinases for spacer sequence specificity, offering a novel concept to enhance the applied properties of designer-recombinases for genome engineering applications.

ORGANISM(S): Escherichia coli

PROVIDER: GSE254392 | GEO | 2024/07/25

REPOSITORIES: GEO

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Chd4 controls Ripk3 dependent necroptosis during muscle regeneration

Project description:We performed a label-free LC-MS/MS study to analyse the role of Chd4 regulation in MuSC in suppressing genes enabling programmed cell death as part of the muscle regeneration program. Lox-cre recombination was used to create a deletion mutant of the CHD4 chromatin modifier (exons 12-21). A pseudo "infection" with GFP was used as control. Proteomics analysis here serves as a method to compare the differentially deregulated proteins upon knockdown of Chd4 in MuSCs using Adenoviral CRE recombination.

2020-04-02 | PXD010370 | Pride

Lamin A/C ablation in pancreatic acinar cells

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2017-11-30 | GSE97396 | GEO

Gene expression profile in the small Maf-deficient livers.

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Genome-wide analysis of gene expression of Flk1Cre;ETV2 CKO mouse yolk sacs.

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Dual leucine zipper kinase is required for excitotoxicity induced neuronal degeneration

Project description:To investigate the role of DLK in the adult brain, we generated mice in which excision of the DLK allele can be temporally controlled to avoid the lethality observed at early postnatal ages in DLK null mice (Hirai et al., 2006). This was done by crossing mice with DLK exons 2-5 flanked by loxP sites (DLKlox/lox) to a Tamoxifen inducible Cre-ERT transgene driven by the CMV early enhancer/chicken beta actin (CAG) promoter that expresses high levels of Cre-ERT in brain and most peripheral tissues (Hayashi and McMahon, 2002). DLKlox/lox /Cre-ERTpos mice (referred to as DLKlox;Crepos) displayed minimal recombination of the DLK allele in the absence of Tamoxifen and survived to adulthood. To induce DLK recombination, 10-12 week old DLKlox;Crepos mice were put on a Tamoxifen diet for three weeks combined with three high dose injections of Tamoxifen, which resulted in efficient excision of DLK in most brain regions. Although the vast majority of DLK protein was eliminated in many brain regions one week after completion of Tamoxifen dosing, a small amount of DLK protein was still present, consistent with the levels of unrecombined DLK observed by PCR in each brain region. Nonetheless, this dosing regimen achieved a reduction in DLK levels that was adequate to assess the function of this kinase in the adult CNS and thus, avoid confounding developmental phenotypes. To examine what changes in gene expression resulted from Tamoxifen induced excision of DLK, we performed microarray analysis on the hippocampi of five Tamoxifen treated DLKlox;Crepos (conditional knock-outs, cKO) and five DLKlox;Creneg (WT) aged matched controls. Gene expression of adult hippocampus was compared betwween DLK wild-type and DLK conditional knockout mice.

2013-08-28 | E-GEOD-50245 | biostudies-arrayexpress

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2020-01-01 | GSE140655 | GEO

Intra-Vk Cluster Recombination Shapes the Ig Kappa Locus Repertoire [END-seq]

2020-01-01 | GSE140635 | GEO

Intra-Vk Cluster Recombination Shapes the Ig Kappa Locus Repertoire [HTGTS]

2020-01-01 | GSE140672 | GEO

RNA-seq of Bifidobacterium longum NCC2705

Project description:Bacterial promoters consist of core sequence motifs termed –35 and –10 boxes. The consensus motifs are TTGACA and TATAAT, respectively, which were identified from leading investigations on E. coli. However, the consensus sequences are not likely to fit genetically divergent bacteria. The sigma factor of the genus Bifidobacterium has a characteristic polar domain in the N-terminus, suggesting the possibility of specific promoter recognition. We reevaluated the structure of B. longum NCC2705 promoters and compared them to other bacteria. Transcriptional start sites (TSS) of the B. longum NCC2705 strain were identified using RNA-Seq analysis to extract promoter regions. Conserved motifs of a bifidobacterial promoter were determined using regions upstream of TSSs and a hidden Markov model. As a result, consensus motifs of the –35 and –10 boxes were TTGTGC and TACAAT, respectively. To assess each base of both motifs, we constructed thirty-seven plasmids based on pKO403-TPCTcon, including the hup promoter connected with a chloramphenicol acetyltransferase as a reporter gene. This reporter assay showed two optimal motifs of the –35 and –10 boxes, TTGNNN and TANNNT, respectively. We further analyzed spacer-lengths between the –35 and –10 boxes via a bioinformatics approach. The spacer-lengths predominant in bacteria have been generally reported to be approximately 17 bp. In contrast, the predominant spacer-lengths in the genus Bifidobacterium and related species were 11 bp, in addition to 17 bp. A reporter assay to assess the spacer-lengths indicated that the 11 bp spacer-length produced unusually high activity.

2020-01-26 | GSE143410 | GEO

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2013-08-28 | GSE50245 | GEO

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