Genome modifications in plant cells by custom-made restriction enzymes

Genome editing, i.e. the ability to mutagenize, insert, delete and replace sequences, in living cells is a powerful and highly desirable method that could potentially revolutionize plant basic research and applied biotechnology. Indeed, various research groups from academia and industry are in a race to devise methods and develop tools that will enable not only site-specific mutagenesis but also controlled foreign DNA integration and replacement of native and transgene sequences by foreign DNA, in living plant cells. In recent years, much of the progress seen in gene targeting in plant cells has been attributed to the development of zinc finger nucleases and other novel restriction enzymes for use as molecular DNA scissors. The induction of double-strand breaks at specific genomic locations by zinc finger nucleases and other novel restriction enzymes results in a wide variety of genetic changes, which range from gene addition to the replacement, deletion and site-specific mutagenesis of endogenous and heterologous genes in living plant cells. In this review, we discuss the principles and tools for restriction enzyme-mediated gene targeting in plant cells, as well as their current and prospective use for gene targeting in model and crop plants.     

A toolbox and procedural notes for characterizing novel zinc finger nucleases for genome editing in plant cells

The induction of double-strand breaks (DSBs) in plant genomes can lead to increased homologous recombination or site-specific mutagenesis at the repair site. This phenomenon has the potential for use in gene targeting applications in plant cells upon the induction of site-specific genomic DSBs using zinc finger nucleases (ZFNs). Zinc finger nucleases are artificial restriction enzymes, custom-designed to cleave a specific DNA sequence. The tools and methods for ZFN assembly and validation could potentially boost their application for plant gene targeting. Here we report on the design of biochemical and in planta methods for the analysis of newly designed ZFNs. Cloning begins with de novo assembly of the DNA-binding regions of new ZFNs from overlapping oligonucleotides containing modified helices responsible for DNA-triplet recognition, and the fusion of the DNA-binding domain with a Fok I endonuclease domain in a dedicated plant expression cassette. Following the transfer of fully assembled ZFNs into Escherichia coli expression vectors, bacterial lysates were found to be most suitable for in vitro digestion analysis of palindromic target sequences. A set of three in planta activity assays was also developed to confirm the nucleic acid digestion activity of ZFNs in plant cells. The assays are based on the reconstruction of GUS expression following transient or stable delivery of a mutated uidA and ZFN-expressing cassettes into target plants cells. Our tools and assays offer cloning flexibility and simple assembly of tested ZFNs and their corresponding target sites into Agrobacterium tumefaciens binary plasmids, allowing efficient implementation of ZFN-validation assays in planta .     

Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage

Custom-designed zinc finger nucleases (ZFNs) are becoming powerful tools in gene targeting-the process of replacing a gene within a genome by homologous recombination. Here, we have studied the DNA cleavage by one such ZFN, DeltaQNK-FN, in order to gain insight into how ZFNs cleave DNA and how two inverted sites promote double-strand cleavage. DNA cleavage by DeltaQNK-FN is greatly facilitated when two DeltaQNK-binding sites are close together in an inverted orientation. Substrate cleavage was not first order with respect to the concentration of DeltaQNK-FN, indicating that double-strand cleavage requires dimerization of the FokI cleavage domain. Rates of DNA cleavage decrease as the substrate concentrations increase, suggesting that the DeltaQNK-FN molecules are effectively "trapped" in a 1:1 complex on DNA when the DNA is in excess. The physical association of two ZFN monomers on DNA was monitored by using the biotin-pull-down assay, which showed that the formation of DeltaQNK-FN active complex required both binding of the two DeltaQNK-FN molecules to specific DNA sites and divalent metal ions.     

Targeted disruption of exogenous EGFP gene in medaka using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are artificial enzymes that create site-specific double-strand breaks and thereby induce targeted genome editing. Here, we demonstrated successful gene disruption in somatic and germ cells of medaka (Oryzias latipes) using ZFN to target exogenous EGFP genes. Embryos that were injected with an RNA sequence pair coding for ZFNs showed mosaic loss of green fluorescent protein fluorescence in skeletal muscle. A number of mutations that included both deletions and insertions were identified within the ZFN target site in each embryo, whereas no mutations were found at the non-targeted sites. In addition, ZFN-induced mutations were introduced in germ cells and efficiently transmitted to the next generation. The mutation frequency varied (6-100%) in the germ cells from each founder, and a founder carried more than two types of mutation in germ cells. Our results have introduced the possibility of targeted gene disruption and reverse genetics in medaka.     

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of ¹³⁷Cs γ-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut

Genome modification by homology‐directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR‐mediated gene exchange of expression cassettes in tobacco BY‐2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7‐kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4‐kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR‐mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin‐resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN‐based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants.     

53BP1: A key player of DNA damage response with critical functions in cancer

Maintenance of genome integrity and stability is a critical responsibility of the DNA damage response (DDR) within cells, such that any disruption in this kinase-based signaling pathway leads to development of various disorders, particularly cancer. The tumor suppressor P53-binding protein 1 (53BP1), as one of the main mediators of DDR, plays a pivotal role in orchestrating the choice of double-strand break (DSB) repair pathway and contains interaction surfaces for numerous DSB-responsive proteins. It has been extensively demonstrated that aberrant expression of 53BP1 contributes to tumor occurrence and development. 53BP1 loss of function in tumor tissues is also related to tumor progression and poor prognosis in human malignancies. Due to undeniable importance of this protein in various aspects of cancer initiation/progression, angiogenesis, metastasis and development of drug resistance, as well as its targeting in the treatment of cancer, this review focused on explaining the structure and function of 53BP1 and its contribution to cancer.     

A reporter mouse for in vivo detection of DNA damage in embryonic germ cells

Maintaining genome integrity in the germline is essential for survival and propagation of a species. In both mouse and human, germ cells originate during fetal development and are hypersensitive to both endogenous and exogenous DNA damaging agents. Currently, mechanistic understanding of how primordial germ cells respond to DNA damage is limited in part by the tools available to study these cells. We developed a mouse transgenic reporter strain expressing a 53BP1‐mCherry fusion protein under the control of the Oct4ΔPE embryonic germ cell‐specific promoter. This reporter binds sites of DNA double strand breaks (DSBs) on chromatin, forming foci. Using ionizing radiation as a DNA DSB‐inducing agent, we show that the transgenic reporter expresses specifically in the embryonic germ cells of both sexes and forms DNA damage induced foci in both a dose‐ and time‐dependent manner. The dynamic time‐sensitive and dose‐sensitive DNA damage detection ability of this transgenic reporter, in combination with its specific expression in embryonic germ cells, makes it a versatile and valuable tool for increasing our understanding of DNA damage responses in these unique cells.     

Functional analyses of the Dof domain, a zinc finger DNA-binding domain, in a pumpkin DNA-binding protein AOBP

AOBP, a DNA-binding protein in pumpkin, contains a Dof domain that is composed of 52 amino acid residues and is highly conserved in several DNA-binding proteins of higher plants. The Dof domain has a significant resemblance to Cys2/Cys2 zinc finger DNA-binding domains of steroid hormone receptors and GATA1, but has a longer putative loop where an extra Cys residue is conserved. We show that the Dof domain in AOBP functions as a zinc finger DNA-binding domain and suggest that the Cys residue uniquely conserved in the putative loop might negatively regulate the binding to DNA.     

The mechanism of extrachromosomal homologous DNA recombination in plant cells

Summary By cotransfecting plasmids carrying particular mutations in the -glucuronidase (GUS) gene into Nicotiana plumbaginifolia protoplasts and by monitoring the recombination rates using a recently developed transient assay, we were able to obtain insights into the mechanism of extrachromosomal recombination operating in plant cells. An exchange of flanking markers takes place in over 90% of the recombination events. In most of the remaining cases two consecutive, independent single crossover events occur. These events involve the same DNA substrate and lead to two successive exchanges of flanking markers, thus mimicking a presumed double crossover intermediate. A comparison of the outcome of our experiments with the predictions of two recombination models originally proposed for mammalian cells indicates that extrachromosomal recombination in plant cells is best described by the single strand annealing model. According to this model all recombination events result in an exchange of flanking markers. Our results rule out the double strand break repair model which predicts that flanking markers are exchanged in only half of all events.     

DNA single stranded gaps formed during DNA repair synthesis induced by methyl methanesulfonate are filled by sequential action of aphidicolin- and dideoxythymidine sensitive DNA polymerases in HeLa cells

DNA repair synthesis induced by methyl methanesulfonate in preconditioned HeLa cells in which DNA replicative synthesis had been highly suppressed was inhibited by aphidicolin (an inhibitor of DNA polymerases and ) and dideoxythymidine (ddThR, an inhibitor of DNA polymerase ). Incomplete repair patches sensitive to exonuclease III were accumulated in the presence of aphidicolin while not in the presence of ddThR. These patches were comopleted by the combined action of Klenow fragment and T4 DNA ligase, indicating that the single-stranded gaps were formed during the repair synthesis. Moreover, ddThR had little effect on the repair synthesis in the presence of aphidicolin. Thus, the results suggest that the single-stranded gaps may be sealed first by aphidicolin-sensitive polymerase followed by ddThR-sensitive DNA polymerase on the same site of the repair patch.Abbreviations ddThR dideoxythymidine - MMS methyl methanesulfonate - dNTP deoxynucleoside triphosphate     

Human somatic cells deficient for RAD52 are impaired for viral integration and compromised for most aspects of homology-directed repair

Homology-directed repair (HDR) maintains genomic integrity by eliminating lesions such as DNA double-strand breaks (DSBs), interstrand crosslinks (ICLs) and stalled replication forks and thus a deficiency in HDR is associated with genomic instability and cancer predisposition. The mechanism of HDR is best understood and most rigorously characterized in yeast. The inactivation of the fungal radiation sensitive 52 (RAD52) gene, which has both recombination mediator and single-strand annealing (SSA) activities in vitro, leads to severe HDR defects in vivo. Confusingly, however, the inactivation of murine and chicken RAD52 genes resulted in mouse and chicken cells, respectively, that were largely aphenotypic. To clarify this issue, we have generated RAD52 knockout human cell lines. Human RAD52-null cells retain a significant level of SSA activity demonstrating perforce that additional SSA-like activities must exist in human cells. Moreover, we confirmed that the SSA activity associated with RAD52 is involved in, but not absolutely required for, most HDR subpathways. Specifically, a deficiency in RAD52 impaired the repair of DNA DSBs and intriguingly decreased the random integration of recombinant adeno-associated virus (rAAV). Finally, an analysis of pan-cancer genome data from The Cancer Genome Atlas (TCGA) revealed an association between aberrant levels of RAD52 expression and poor overall survival in multiple cancers. In toto, our work demonstrates that RAD52 contributes to the maintenance of genome stability and tumor suppression in human cells.     

Site-specific integration of retroviral DNA in human cells using fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc-finger protein E2C

During the life cycle of retroviruses, establishment of a productive infection requires stable joining of a DNA copy of the viral RNA genome into host cell chromosomes. Retroviruses are thus promising vectors for the efficient and stable delivery of genes in therapeutic protocols. Integration of retroviral DNA is catalyzed by the viral enzyme integrase (IN), and one salient feature of retroviral DNA integration is its lack of specificity, as many chromosomal sites can serve as targets for integration. Despite the promise for success in the clinic, one major drawback of the retrovirus-based vector is that any unintended insertion events from the therapy can potentially lead to deleterious effects in patients, as demonstrated by the development of malignancies in both animal and human studies. One approach to directing integration into predetermined DNA sites is fusing IN to a sequence-specific DNA-binding protein, which results in a bias of integration near the recognition site of the fusion partner. Encouraging results have been generated in vitro and in vivo using fusion protein constructs of human immunodeficiency virus type 1 IN and E2C, a designed polydactyl zinc-finger protein that specifically recognizes an 18-base pair DNA sequence. This review focuses on the method for preparing infectious virions containing the IN fusion proteins and on the quantitative PCR assays for determining integration site specificity. Efforts to engineer IN to recognize specific target DNA sequences within the genome may lead to development of effective retroviral vectors that can safely deliver gene-based therapeutics in a clinical setting.     

Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed

Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG^+/+ and UNG^−/− cells. We report that despite identical growth rates and DSB repair capacities, UNG^−/− cells accumulated significantly greater uracil and DSBs compared with UNG^+/+ cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG^+/+ cells. Furthermore, DSBs in UNG^+/+ and UNG^−/− cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG^−/− cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG^+/+ and UNG^−/− cells treated with IC₅₀-level pemetrexed. However, following pemetrexed exposure, UNG^−/− but not UNG^+/+ cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG^−/− cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death.