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Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks.

ABSTRACT: The creation of a DNA break at a specific locus by a designer endonuclease can be harnessed to edit a genome. However, DNA breaks may engage one of several competing repair pathways that lead to distinct types of genomic alterations. Therefore, understanding the contribution of different repair pathways following the introduction of a targeted DNA break is essential to further advance the safety and efficiency of nuclease-induced genome modification. To gain insight into the role of different DNA repair pathways in resolving nuclease-induced DNA breaks into genome editing outcomes, we previously developed a fluorescent-based reporter system, designated the Traffic Light Reporter, which provides a readout of gene targeting and gene disruption downstream of a targeted DNA double-strand break. Here we describe two related but novel reporters that extend this technology: one that allows monitoring of the transcriptional activity at the reporter locus, and thus can be applied to interrogate break resolution at active and repressed loci; and a second that reads out single-strand annealing in addition to gene targeting and gene disruption. Application of these reporters to assess repair pathway usage in several common gene editing contexts confirms the importance that chromatin status and initiation of end resection have on the resolution of nuclease-induced breaks.

SUBMITTER: Kuhar R

PROVIDER: S-EPMC3874187 | biostudies-other | 2014 Jan

REPOSITORIES: biostudies-other

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Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks.

Kuhar Ryan R Gwiazda Kamila S KS Humbert Olivier O Mandt Tyler T Pangallo Joey J Brault Michelle M Khan Iram I Maizels Nancy N Rawlings David J DJ Scharenberg Andrew M AM Certo Michael T MT

Nucleic acids research 20131010 1

The creation of a DNA break at a specific locus by a designer endonuclease can be harnessed to edit a genome. However, DNA breaks may engage one of several competing repair pathways that lead to distinct types of genomic alterations. Therefore, understanding the contribution of different repair pathways following the introduction of a targeted DNA break is essential to further advance the safety and efficiency of nuclease-induced genome modification. To gain insight into the role of different DN ...[more]

PMID: 24121685

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Project description:BackgroundIn this study, we aimed at investigating heterogeneity in the expression of metabolic genes in clonal populations of Escherichia coli growing on glucose as the sole carbon source. Different metabolic phenotypes can arise in these clonal populations through variation in the expression of glucose transporters and metabolic enzymes. First, we focused on the glucose transporters PtsG and MglBAC to analyze the diversity of glucose uptake strategies. Second, we analyzed phenotypic variation in the expression of genes involved in gluconeogenesis and acetate scavenging (as acetate is formed and excreted during bacterial growth on glucose), which can reveal, for instance, phenotypic subpopulations that cross-feed through the exchange of acetate. In these experiments, E. coli MG1655 strains containing different transcriptional GFP reporters were grown in chemostats and reporter expression was measured with flow cytometry.ResultsOur results suggest heterogeneous expression of metabolic genes in bacterial clonal populations grown in glucose environments. The two glucose transport systems exhibited different level of heterogeneity. The majority of the bacterial cells expressed the reporters for both glucose transporters MglBAC and PtsG and a small fraction of cells only expressed the reporter for Mgl. At a low dilution rate, signals from transcriptional reporters for acetyl-CoA synthetase Acs and phosphoenolpyruvate carboxykinase Pck indicated that almost all cells expressed the genes that are part of acetate utilization and the gluconeogenesis pathway, respectively. Possible co-existence of two phenotypic subpopulations differing in acs expression occurred at the threshold of the switch to overflow metabolism. The overflow metabolism results in the production of acetate and has been previously reported to occur at intermediate dilution rates in chemostats with high concentration of glucose in the feed.ConclusionsAnalysis of the heterogeneous expression of reporters for genes involved in glucose and acetate metabolism raises new question whether different metabolic phenotypes are expressed in clonal populations growing in continuous cultures fed on glucose as the initially sole carbon source.

Project description:Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that eliminates a PCR amplifiable target) revealed these cryptic DSBs and facilitated the determination of the true efficacy of genome editing reagents. The results indicated that in HEK293T cells, approximately 40% of the DSBs introduced by genome editing, were available for participation in HDR.

Dataset Information

Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks.

Publications

Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks.

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