Steven R. Bischoff

Base editors are a type of double-stranded break (DSB)-free gene editing technology that has opened up new possibilities for precise manipulation of mitochondrial DNA (mtDNA). This includes cytosine and adenosine base editors and more recently guanosine base editors. Because of having low off-target and indel rates, there is a growing interest in developing and evolving this research field. Here, we provide a detailed update on DNA base editors. While base editing has widely been used for nuclear genome engineering, the growing interest in applying this technology to mitochondrial DNA has been faced with several challenges. While Cas9 protein has been shown to enter mitochondria, use of smaller Cas proteins, such as Cas12a, has higher import efficiency. However, sgRNA transfer into mitochondria is the most challenging step. sgRNA structure and ratio of Cas protein to sgRNA are both important factors for efficient sgRNA entry into mitochondria. In conclusion, while there are still several challenges to be addressed, ongoing research in this field holds the potential for new treatments and therapies for mitochondrial disorders.

The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5′ end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of downstream polyadenylation sequences. Readthrough required Repeat A and the ∼750 nucleotides downstream, did not require SPEN, and was attenuated by splicing. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a 5.5-kb Xist transgene robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5′ end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

Summary: Extracellular vesicles (EVs) are abundant, lipid‐enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type‐specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR‐Cas9‐mediated genome editing to generate mice bearing a CD63‐emGFP^{loxP/stop/loxP} knock‐in cassette that enables the specific labeling of circulating CD63⁺ vesicles from any cell type when crossed with lineage‐specific Cre recombinase driver mice. As proof‐of‐principle, we have crossed these mice with Cdh5‐Cre^ERT2 mice to generate CD63^emGFP+ vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma‐purified EVs contain a subpopulation of emGFP⁺ vesicles that are derived from the endothelium, co‐express additional EV (e.g., CD9 and CD81) and endothelial cell (e.g., CD105) markers, and they harbor specific miRNAs (e.g., miR‐126, miR‐30c, and miR‐125b). This new mouse strain should be a useful genetic tool for generating cell type‐specific, CD63⁺ EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.

Xist requires Repeat-A, a protein-binding module in its first two kilobases (2kb), to repress transcription. We report that when expressed as a standalone transcript in mouse embryonic stem cells (ESCs), the first 2kb of Xist (Xist-2kb) does not induce transcriptional silencing. Instead, Xist-2kb sequesters RNA produced from adjacent genes on chromatin. Sequestration does not spread beyond adjacent genes, requires the same sequence elements in Repeat-A that full-length Xist requires to repress transcription and can be induced by lncRNAs with similar sequence composition to Xist-2kb. We did not detect sequestration by full-length Xist, but we did detect it by mutant forms of Xist with attenuated transcriptional silencing capability. Xist-2kb associated with SPEN, a Repeat-A binding protein required for Xist-induced transcriptional silencing, but SPEN was not necessary for sequestration. Thus, when expressed in mouse ESCs, a 5′ fragment of Xist that contains Repeat-A sequesters RNA from adjacent genes on chromatin and associates with the silencing factor SPEN, but it does not induce transcriptional silencing. Instead, Xist-induced transcriptional silencing requires synergy between Repeat-A and additional sequence elements in Xist. We propose that sequestration is mechanistically related to the Repeat-A dependent stabilization and tethering of Xist near actively transcribed regions of chromatin.

To gain insight into differences in placental physiology between two swine breeds noted for their dissimilar reproductive performance, that is, the Chinese Meishan and white composite (WC), we examined gene expression profiles of placental tissues collected at 25, 45, 65, 85, and 105 days of gestation by microarrays. Using a linear mixed model, a total of 1,595 differentially expressed genes were identified between the two pig breeds using a false-discovery rate q-value ≤0.05. Among these genes, we identified breed-specific isoforms of XIST, a long non-coding RNA responsible X-chromosome dosage compensation in females. Additionally, we explored the interaction of placental gene expression and chromosomal location by DIGMAP and identified three Sus scrofa X chromosomal bands (Xq13, Xq21, Xp11) that represent transcriptionally active clusters that differ between Meishan and WC during placental development. Also, pathway analysis identified fundamental breed differences in placental cholesterol trafficking and its synthesis. Direct measurement of cholesterol confirmed that the cholesterol content was significantly higher in the Meishan versus WC placentae. Taken together, this work identifies key metabolic pathways that differ in the placentae of two swine breeds noted for differences in reproductive prolificacy.

In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing 4 transcription factors, Oct3/4 , Klf4 , Sox2 , and c-Myc (OKSM). Here, we report the derivation of induced pluripotent stem cells (iPSCs) from adult canine fibroblasts by retroviral OKSM transduction. The isolated canine iPSCs (ciPSCs) were expanded in 3 different culture media [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), or FGF2 plus LIF]. Cells cultured in both FGF2 and LIF expressed pluripotency markers [ POU5F1 ( OCT4 ), SOX2 , NANOG , and LIN28 ] and embryonic stem cell (ESC)–specific genes ( PODXL , DPPA5 , FGF5 , REX1 , and LAMP1 ) and showed strong levels of alkaline phosphatase expression. In vitro differentiation by formation of embryoid bodies and by directed differentiation generated cell derivatives of all 3 germ layers as confirmed by mRNA and protein expression. In vivo, the ciPSCs created solid tumors, which failed to reach epithelial structure formation, but expressed markers for all 3 germ layers. Array comparative genomic hybridization and chromosomal fluorescence in situ hybridization analyses revealed that while retroviral transduction per se did not result in significant DNA copy number imbalance, there was evidence for the emergence of low-level aneuploidy during prolonged culture or tumor formation. In summary, we were able to derive ciPSCs from adult fibroblasts by using 4 transcription factors. The isolated iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF2 and LIF are still unknown.

Pluripotent stem cells such as embryonic stem (ES) and iPS cells can give rise to derivatives of all three germ layers and thus have great potential in regenerative medicine. Unfortunately, isolation of ES cells requires availability of embryos. Moreover, it is not possible to genetically match ES cells and patients resulting in immune rejection. iPS cells, in contrast, can be generated from somatic cells from the patients being treated, solving the issue of genetic match and availability of embryos. In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing four transcription factors, Oct3/4, Sox2, Klf4 and c-Myc. Here we report the derivation of putative iPS cells from adult canine fibroblast. VSV-G pseudotyped retroviruses containing transcription factors (Oct3/4, Sox2, Klf4 and c-Myc) were transduced into skin fibroblasts isolated from a 3-year old dog. The transduced cells were cultured in DMEM containing 10% FBS for 6 days. The cells were then replated onto γ–irradiated mouse embryonic fibroblasts (MEFs) and the media was replaced with mTeSR1. A few granulated colonies appeared at day 14 and ES-like colonies were picked at day 21 post-infection. Colonies were expanded in three different ES culture media containing either FGF (10 ng/mL), LIF (10³ units/mL) or both FGF and LIF, and supplemented with two chemical inhibitors (3 μM CHIR99021 and 20 μM PD98059). The isolated canine iPS cells were very similar to dog ES cells in morphology and alkaline phosphatase (AP) expression. Though the morphology and self-renewal of canine iPS cells was maintained over 12 passages in all three conditions, only the cells cultured in (FGF plus LIF) showed strong AP activity. The cells cultured in FGF/LIF were further analyzed by RT-PCR and immunocytochemistry (ICC) and found to express the pluripotency markers, Oct3/4 and Nanog, as well as human ES cell- (Podxl, FGF5 and Rex1) and mouse ES cell- (LAMP1, dppa5 and Brachyury) specific genes. However, the viral transcription factors transduced in the iPS cells were not silenced. Spontaneous differentiation by withdrawal of growth factors led to formation of embryoid bodies (EB) composed of cells representative of all three germ layers as confirmed by both immunostaining and RT-PCR. The EB cells were positive for CXCR4 and Sox17 (endoderm), desmin, vimentin and Nkx2.5 (mesoderm) and GFAP, TuJ1, NCAM and bIII-tubulin (ectoderm). Similar results were seen by directed In vitro differentiation. Subcutaneous injection of the putative canine iPS cells into SCID mice created immature teratomas which failed to reach epithelial structure formation, but expressed markers for all three germ layer as confirmed by both RT-PCR and ICC. In summary, we were able to isolate induced pluripotent cells from cultured adult somatic cells by using four transcription factors. The isolated reprogrammed cells have similar characteristics to ES cells from other species, but the exact cellular mechanisms behind its unique co-dependency on both FGF and LIF is still unknown. Availability of stable canine iPS cells would have significant implications for regenerative medicine in both veterinary and human clinical applications. This work was funded by a grant to JP and NO from the Canine Health Foundation, grant #01272. (poster)