TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus

Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.     

Establishment of a rapid and scalable gene expression system in livestock by site-specific integration

Somatic cell-mediated transgenesis is routinely used to transfer exogenous genes to livestock genomes. However, transgene insertion events are essentially random which may lead to transgene silencing or alter animal phenotype because of insertional mutagenesis. To overcome these problems, we established a gene manipulation system in goat somatic cells based on homologous recombination and flp recombinase-mediated site-specific integration. First, we performed gene targeting to introduce an frt-docking site into the α1 (I) procollagen (ColA1) locus in goat somatic cells. Second, the targeted cell clones were rejuvenated by embryo cloning, and the vigorous cells with targeted frt were reestablished. Third, a gene-replacement system was used to introduce an EGFP reporter gene into the targeted ColA1 locus via flp mediated recombination. As a result, the transgenic somatic cell exhibited faithful expression of EGFP gene under control of the CMV promoter. Similarly, other expression vectors can be introduced into the defined site to evaluate gene functions or express valuable proteins. The gene manipulation system described here will be applicable in other livestock somatic cells, and would allow for the rapid generation of livestock with transgene targeted to the defined site.     

The isolation and characterization of nrc-1 and nrc-2, two genes encoding protein kinases that control growth and development in Neurospora crassa.

Using an insertional mutagenesis approach, a series of Neurospora crassa mutants affected in the ability to control entry into the conidiation developmental program were isolated. One such mutant, GTH16-T4, was found to lack normal vegetative hyphae and to undergo constitutive conidiation. The affected gene has been named nrc-1, for nonrepressible conidiation gene #1. The nrc-1 gene was cloned from the mutant genomic DNA by plasmid rescue, and was found to encode a protein closely related to the protein products of the Saccharomyces cerevisiae STE11 and Schizosaccharomyces pombe byr2 genes. Both of these genes encode MAPKK kinases that are necessary for sexual development in these organisms. We conclude the nrc-1 gene encodes a MAPKK kinase that functions to repress the onset of conidiation in N. crassa. A second mutant, GTH16-T17, was found to lack normal vegetative hyphae and to constitutively enter, but not complete, the conidiation program. The affected locus is referred to as nrc-2 (nonrepressible conidiation gene #2). The nrc-2 gene was cloned and found to encode a serine-threonine protein kinase. The kinase is closely related to the predicted protein products of the S. pombe kad5, and the S. cerevisiae YNRO47w and KIN82 genes, three genes that have been identified in genome sequencing projects. The N. crassa nrc-2 gene is the first member of this group of kinases for which a phenotype has been defined. We conclude a functional nrc-2-encoded serine/threonine kinase is required to repress entry into the conidiation program.     

Molecular analysis of mutants of the <Emphasis Type="Italic">Neurospora</Emphasis> adenylosuccinate synthetase locus

The ad-8 gene of Neurospora crassa, in addition to being used for the study of purine biology, has been extensively studied as a model for gene structure, mutagenesis and intralocus recombination. Because of this there is an extensive collection of well-characterized N. crassa ad-8 mutants in the Fungal Genetics Stock Center collection. Among these are spontaneous mutants and mutants induced with X-ray, UV or chemical mutagens. The specific lesions in these mutants have been genetically mapped at high resolution. We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature of the mutation in each strain. We compare the historical fine-structure map to the DNA and amino acid sequence of each allele. The placement of the individual lesions in the fine-structure map was more accurate at the 5' end of the gene and no mutants were identified in the 3' untranslated region of this gene. We additionally analysed ad-8(+) alleles in 18 N. crassa strains subjected to whole-genome sequence analysis and describe the variability among Neurospora strains and among fungi and other organisms.     

Targeting vectors for gene diversification by meiotic recombination in Neurospora crassa.

We have constructed a pair of vectors, pDV2 and pDV3, that enable targeted insertion of exogenous DNA into Linkage Group I of Neurospora crassa at the his-3 locus. Transplaced sequences are inserted between his-3 and the cog(L) recombination hot spot and include his-3 mutations that allow meiotic recombination initiated by cog(L) to be monitored. Selection of correctly placed transforming DNA is based on complementation between different his-3 alleles borne by the plasmids and transformation hosts. The system allows investigation of the effect of any given sequence on recombination as well as diversification of sets of related sequences in vivo for directed evolution of genes.     

Generation of single-copy T-DNA transformants in Arabidopsis by the CRE/loxP recombination-mediated resolution system

We investigated whether complex T-DNA loci, often resulting in low transgene expression, can be resolved efficiently into single copies by CRE/loxP-mediated recombination. An SB-loxP T-DNA, containing two invertedly oriented loxP sequences located inside and immediately adjacent to the T-DNA border ends, was constructed. Regardless of the orientation and number of SB-loxP-derived T-DNAs integrated at one locus, recombination between the outermost loxP sequences in direct orientation should resolve multiple copies into a single T-DNA copy. Seven transformants with a complex SB-loxP locus were crossed with a CRE-expressing plant. In three hybrids, the complex T-DNA locus was reduced efficiently to a single-copy locus. Upon segregation of the CRE recombinase gene, only the simplified T-DNA locus was found in the progeny, demonstrating DNA had been excised efficiently in the progenitor cells of the gametes. In the two transformants with an inverted T-DNA repeat, the T-DNA resolution was accompanied by at least a 10-fold enhanced transgene expression. Therefore, the resolution of complex loci to a single-copy T-DNA insert by the CRE/loxP recombination system can become a valuable method for the production of elite transgenic Arabidopsis thaliana plants that are less prone to gene silencing.     

Cytogenetic Localization of the Acid Phosphatase-1 Gene in DROSOPHILA MELANOGASTER

A translocation in which a segment of chromosome 3 is inserted into the Y chromosome was found to contain the acid phosphatase-1 gene (Acph-1). In flies hyperploid for that gene, acid phosphatase-1 levels are proportional to the dose of the gene. The locus is placed within the salivary chromosome subdivisions 99D and 99E on the basis of its inclusion in the translocated segment and on the previous placement of the claret locus. Several chromosomal rearrangements involving heterochromatic breakpoints and euchromatic breakpoints adjacent to 99D-99E were tested for possible postiion-effect variegation of acid phosphatase-1. No decrease in the synthesis of the electorphoretic subunit encoded by the relocated gene was observed within any of the rearrangements.     

Cloning the quinic acid (aq) gene cluster from Neurospora crassa: identification of recombinant plasmids containing both qa-2+ and qa-3+

A 22.2-kb insert of Neurospora crassa DNA containing at least two of the genes from the inducible catabolic quinic acid pathway has been cloned into the cosmid vehicle pHC79 resulting in a recombinant plasmid, pMSK308. The qa-2+ locus (which encodes catabolic dehydroquinase) is functionally expressed in both Escherichia coli and qa-2 mutants of N. crassa transformed with pMSK308 plasmid DNA. Expression of the qa-3 gene (which encodes quinate dehydrogenase) is only detected upon reintroduction into N. crassa. Results were also obtained which suggested that the qa-4 gene, which maps between qa-2 and qa-3, may also be present on both pMSK308 and the previously described plasmid pVK88. Certain anomalies in the types of N. crassa transformants obtained with pMSK308 plasmid DNA were noted.     

Molecular Cloning of a Gene (Cfp) Encoding the Cytoplasmic Filament Protein P59nc and Its Genetic Relationship to the Snowflake Locus of Neurospora Crassa

P59Nc is a 59-kD polypeptide associated with 8-10-nm diameter cellular filaments in normal Neurospora crassa strains. Abnormally sized and shaped bundles of these structures are present in N. crassa strains carrying mutations at the locus sn (snowflake). By using molecular cloning and restriction fragment length polymorphism (RFLP) segregation analysis strategies we show here that sn is not the genetic locus of P59Nc. Several P59Nc cDNAs were cloned from a N. crassa lambda GT11 library after immunoscreening with specific polyclonal anti-P59Nc antibodies. Additional longer cDNAs were obtained from a N. crassa cDNA-lambda ZAP library. When used as probes in Southern blots of total DNA from wild-type strains, multicent-2 (a multiple mutant strain), and snowflake mutants, the P59Nc cDNAs revealed comparable patterns of hybridizing bands for all of the restriction enzymes tested. Analysis of segregation of BclI and ClaI RFLPs, detected in the genomic region of the P59Nc gene (locus cfp: cellular filament polypeptide), among a set of strains designed for RFLP mapping, or among selected progeny of crosses involving a snowflake parent, respectively, indicate that (i) there is in N. crassa a single cfp locus positioned on the right arm of linkage group VII between the locus for and the proximal breakpoint of the translocation T(VII----I)5936; (ii) the sn mutations in the centromere region of chromosome I do not represent translocations of cfp; and (iii) the snowflake mutants possesses a normal copy of the P59Nc gene on their chromosomes VII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci.

Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance of ADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus.     

Neurospora crassa ve-1 affects asexual conidiation

The velvet factor of the homothallic fungus Aspergillus nidulans promotes sexual fruiting body formation. The encoding veA gene is conserved among fungi, including the ascomycete Neurospora crassa. There, the orthologous ve-1 gene encodes a deduced protein with high similarity to A. nidulans VeA. Cross-complementation experiments suggest that both the promoter and the coding sequence of N. crassa ve-1 are functional to complement the phenotype of an A. nidulans deletion mutant. Moreover, ve-1 expression in the heterologous host A. nidulans results in development of reproductive structures in a light-dependent manner, promoting sexual development in the darkness while stimulating asexual sporulation under illumination. Deletion of the N. crassa ve-1 locus by homologous gene replacement causes formation of shortened aerial hyphae accompanied by a significant increase in asexual conidiation, which is not light-dependent. Our data suggest that the conserved velvet proteins of A. nidulans and N. crassa exhibit both similar and different functions to influence development of these two ascomycetes.     

Cyclosporin A-binding protein (cyclophilin) of Neurospora crassa. One gene codes for both the cytosolic and mitochondrial forms

Cyclophilin (cyclosporin A-binding protein) has a dual localization in the mitochondria and in the cytosol of Neurospora crassa. The two forms are encoded by a single gene which is transcribed into mRNAs having different lengths and 5' termini (approximately 1 and 0.8 kilobases). The shorter mRNA specifies the cytosolic protein consisting of 179 amino acids. The longer mRNA is translated into a precursor polypeptide with an amino-terminal extension of 44 amino acids which is cleaved in two steps upon entry into the mitochondrial matrix. Neurospora cyclophilin shows about 60% sequence homology to human and bovine cyclophilins.     

Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1.

Molybdopterin (MPT) is not produced by the Escherichia coli mutants chlA1, chlM, or chlN or by the Neurospora crassa mutant nit-1. Extracts of E. coli chlA1 contain an activity, the converting factor, which is functionally defined by its ability to convert a low-molecular-weight precursor present in crude extracts of N. crassa nit-1 into molybdopterin in vitro. In this study, it has been shown that the converting factor consists of two associative proteins (10 and 25 kilodaltons [kDa]) which can be separated by using either anion-exchange or gel filtration chromatography. Neither protein is able to complement extracts of nit-1 by itself. Analysis of chlA Mu insertion mutants has shown that the two proteins are distinct gene products encoded at the chlA locus. Twelve chlA Mu insertion strains which lacked converting factor activity were deficient in one or both of the proteins. Converting factor activity could be generated by mixing extracts from strains having the 25-kDa protein with those having the 10-kDa protein but not those lacking both proteins. Finally, it was shown that the chlM mutant lacks the 10-kDa protein while the chlN mutant, which contains both the 10- and 25-kDa proteins, lacks a function required to activate the 10-kDa protein.     

Knockout targeting of the Drosophila nap1 gene and examination of DNA repair tracts in the recombination products

We used ends-in gene targeting to generate knockout mutations of the nucleosome assembly protein 1 (Nap1) gene in Drosophila melanogaster. Three independent targeted null-knockout mutations were produced. No wild-type NAP1 protein could be detected in protein extracts. Homozygous Nap1(KO) knockout flies were either embryonic lethal or poorly viable adult escapers. Three additional targeted recombination products were viable. To gain insight into the underlying molecular processes we examined conversion tracts in the recombination products. In nearly all cases the I-SceI endonuclease site of the donor vector was replaced by the wild-type Nap1 sequence. This indicated exonuclease processing at the site of the double-strand break (DSB), followed by replicative repair at donor-target junctions. The targeting products are best interpreted either by the classical DSB repair model or by the break-induced recombination (BIR) model. Synthesis-dependent strand annealing (SDSA), which is another important recombinational repair pathway in the germline, does not explain ends-in targeting products. We conclude that this example of gene targeting at the Nap1 locus provides added support for the efficiency of this method and its usefulness in targeting any arbitrary locus in the Drosophila genome.