Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice

ABSTRACTCRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as concatemers. Here, we show that injecting 5’biotinylated donor DNA in mouse embryos at the two-cell stage leads to efficient single-copy HDR (scHDR) alleles. Our dedicated genotyping strategy showed that these alleles occurred with a frequency of 19%, 20%, and 26%, respectively, in three independent gene loci, indicating that scHDR is dramatically boosted by 5’biotinylation. Thus, we suggest that a combination of a 5’biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins into the mouse genome.

In-planta Gene Targeting in Barley Using Cas9 With and Without Geminiviral Replicons

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications; however, this remains inefficient especially in key crop species. Here, we describe successful, heritable gene targeting in barley at the target site of Cas9 using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase the copy number of the repair template. Without the replicon, we were able to delete 150 bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared with the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false-positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1, and subsequently, the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

Are Cyanobacteria an Ancestor of Chloroplasts or Just One of the Gene Donors for Plants and Algae?

Chloroplasts of plants and algae are currently believed to originate from a cyanobacterial endosymbiont, mainly based on the shared proteins involved in the oxygenic photosynthesis and gene expression system. The phylogenetic relationship between the chloroplast and cyanobacterial genomes was important evidence for the notion that chloroplasts originated from cyanobacterial endosymbiosis. However, studies in the post-genomic era revealed that various substances (glycolipids, peptidoglycan, etc.) shared by cyanobacteria and chloroplasts are synthesized by different pathways or phylogenetically unrelated enzymes. Membranes and genomes are essential components of a cell (or an organelle), but the origins of these turned out to be different. Besides, phylogenetic trees of chloroplast-encoded genes suggest an alternative possibility that chloroplast genes could be acquired from at least three different lineages of cyanobacteria. We have to seriously examine that the chloroplast genome might be chimeric due to various independent gene flows from cyanobacteria. Chloroplast formation could be more complex than a single event of cyanobacterial endosymbiosis. I present the “host-directed chloroplast formation” hypothesis, in which the eukaryotic host cell that had acquired glycolipid synthesis genes as an adaptation to phosphate limitation facilitated chloroplast formation by providing glycolipid-based membranes (pre-adaptation). The origins of the membranes and the genome could be different, and the origin of the genome could be complex.

Molecular Background of Estrogen-Dependent and -Independent Gene Expressions in Endometriotic Lesions

Background: Endometriosis is an estrogen-dependent disease, and the role of estrogen is obvious because the symptoms associated with endometriosis often disappear after menopause, and GnRH agonists or progestin relieve the pelvic lesions and endometriosis-associated pain. However, there are limitations to these treatments that target the estrogen reduction in endometriotic lesions. As a possible background, we hypothesized a role of the local environment with high estrogen depending on aromatase upregulation in endometriotic lesions. Objective: To test our hypothesis, we re-evaluated the expression profile of estrogen receptor (ER), and then searched for the estrogen-dependent gene expressions in endometriotic cells. Finally, we approached the epigenetic background of gene expressions in endometriotic cells. Patients: Institutional Review Boards approved this project. We obtained the informed consent from all patients. The chocolate cyst lining in ovaries of patients with endometriosis was the source of endometriotic tissue. As the control, the eutopic endometrial tissues were obtained from uteri of premenopausal women who had uterine leiomyoma. Methods: Stromal cells were prepared from endometriotic and endometrial tissues. Gene expression was evaluated using RT-PCR. Specific primer sets of unique 5’-UTR exons/exon 2 in ESR1 and specific primer sets of unique 5’-UTR exons/exon1 in ESR2 were used for the analysis of promoter usage. Primer sets of exon 7 and exon 8 in ESR2 were used to evaluate the expression of ERβisoform. Using SERM(PPT and DPN), ER-dependent gene expression was estimated. The potential function of hypomethylated gene sequence as an active enhancer was evaluated by ChIP analysis and eRNA expression. Results: 1) Relative expression of ERα mRNA in endometriotic cells was estimated to be one tenth of that in endometrial cells. 2) Relative expression of ERβ1 mRNA was 40-fold higher than that in endometrial cells, which is almost at a comparable level of the ERα. 3) In addition to ERβ1 mRNA, a splice variant ERβ2 was expressed at a comparable level of the ERβ1. 4) Top ten genes, up- or down-regulated in response to SERM, were extracted in endometriotic cells. 5) TGFA expression was upregulated at a comparable level in response to PPT and DPN. 6) A stretch of hypomethylated sequence, which includes an ERE at 50kb upstream from the TSS, was suggested as active enhancer. 7) ESR1 and ESR2showed a marginal response to SERM. 8) GATA6 and CYP19 were highly expressed in endometriotic cells, and hypomethylated sequences in these genes were suggested as active enhancer. Conclusion: In the hope of overcoming the limitations of endocrine treatments in endometriosis, we examined ER-dependent and -independent gene expressions using endometriotic cells. The results suggest one aspect of gene expression in endometriosis lesions.

Identification of the Roles of a Stemness Index Based on mRNA Expression in the Prognosis and Metabolic Reprograming of Pancreatic Ductal Adenocarcinoma

BackgroundCancer stem cells (CSCs) are widely thought to contribute to the dismal prognosis of pancreatic ductal adenocarcinoma (PDAC). CSCs share biological features with adult stem cells, such as longevity, self-renewal capacity, differentiation, drug resistance, and the requirement for a niche; these features play a decisive role in cancer progression. A prominent characteristic of PDAC is metabolic reprogramming, which provides sufficient nutrients to support rapid tumor cell growth. However, whether PDAC stemness is correlated with metabolic reprogramming remains unknown.MethodRNA sequencing data of PDAC, including read counts and fragments per kilobase of transcript per million mapped reads (FPKM), were collected from The Cancer Genome Atlas-Pancreatic Adenocarcinoma (TCGA-PAAD) database. Single-sample gene set enrichment analysis (GSEA) was used to calculate the relative activities of metabolic pathways in each PDAC sample. Quantitative real-time PCR was performed to validate the expression levels of genes of interest.ResultsThe overall survival (OS) of patients with high mRNA expression-based stemness index (mRNAsi) values was significantly worse than that of their counterparts with low mRNAsi values (P = 0.003). This survival disadvantage was independent of baseline clinical characteristics. Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and GSEA showed that the differentially expressed genes between patients with high and low mRNAsi values were mainly enriched in oncogenic and metabolic pathways. Weighted gene coexpression network analysis (WGCNA) revealed 8 independent gene modules that were significantly associated with mRNAsi and 12 metabolic pathways. Unsupervised clustering based on the key genes in each module identified two PDAC subgroups characterized by different mRNAsi values and metabolic activities. Univariate Cox regression analysis identified 14 genes beneficial to OS from 95 key genes selected from the eight independent gene modules from WGCNA. Among them, MAGEH1, MAP3K3, and PODN were downregulated in both pancreatic tissues and cell lines.ConclusionThe present study showed that PDAC samples with high mRNAsi values exhibited aberrant activation of multiple metabolic pathways, and the patients from whom these samples were obtained had a poor prognosis. Future studies are expected to investigate the underlying mechanism based on the crosstalk between PDAC stemness and metabolic rewiring.

In-planta Gene Targeting in Barley using Cas9, with and without Geminiviral Replicons

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications, however this remains inefficient especially in key crop species. Here we describe successful, heritable gene targeting in barley using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase copy number of the repair template. Without the replicon, we were able to delete 150bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared to the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1 and subsequently the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

Independent Pseudogenizations and Losses of Sox15 During Amniote Diversification Following Asymmetric Ohnolog Evolution

Background: Four ohnologous genes (sox1, sox2, sox3, and sox15) were generated by two rounds of wholegenome duplication in a vertebrate ancestor. In eutherian mammals, Sox1, Sox2, and Sox3 participate in central nervous system (CNS) development. Sox15 functions in skeletal muscle regeneration, and has little functional overlap with the other three ohnologs. In contrast, Xenopus frog and zebrafish orthologs of sox15 as well as sox1-3s are expressed and function in CNS development. We previously reported that Sox15 is involved in mouse placental development as neofunctionalization, but is pseudogenized in marsupial opossum. These findings suggest that sox15 might have evolved with unusual gene fates during vertebrate evolution. However, knowledge concerning sox15 in other vertebrate lineages is scant. Our purpose was to clarify the fate and molecular evolution of sox15 during vertebrate evolution.Results: We searched for sox15 orthologs in various vertebrate lineages by homology and synteny analyses using vertebrate genome databases. Interestingly, sox15 was independently pseudogenized at least twice during species diversity in marsupial mammals. Moreover, we observed independent gene loss of sox15 at least twice during reptile evolution in squamates and crocodile-bird diversification. Codon-based phylogenetic tree and selective analyses revealed the highest dN/dS value for sox15 among the four ohnologs during jawed vertebrate evolution. The finding was supported by the high values in cartilaginous fishes, anuran amphibians, and amniotes. The high dN/dS value of sox15 may have been mainly caused by a relaxed selection. Marsupial and squamate sox15 may have evolved under more relaxed selection than those of eutherian mammals and testudine reptiles, respectively. Conclusions: The findings revealed an asymmetric evolution of sox15 among the four ohnologs during vertebrate evolution. Notably, independent pseudogenizations and losses of sox15 were observed during marsupial and reptile evolution, respectively. Both might have been caused by strong relaxed selection. The drastic gene fates of sox15, including neofunctionalization and pseudogenizations/losses during amniote diversification, might be caused by a release from evolutionary constraints. We discuss why sox15 has evolved under relaxed selection, considering the possible escapes from some constraints there could have been during its molecular evolution.