Gene editing of the mitochondrial genome using the CRISPR-Cas9 system is highly challenging mainly due to sub-efficient delivery of guide RNA and Cas9 enzyme complexes into the mitochondria. In this study, we were able to perform gene editing in the mitochondrial DNA by appending an NADH-ubiquinone oxidoreductase chain 4 (ND4) targeting guide RNA to an RNA transport-derived stem loop element (RP-loop) and expressing the Cas9 enzyme with a preceding mitochondrial localization sequence. We observe mitochondrial colocalization of RP-loop gRNA and a marked reduction of ND4 expression in the cells carrying a 11205G variant in their ND4 sequence coincidently decreasing the mtDNA levels. This proof-of-concept study suggests that a stem-loop element added sgRNA can be transported to the mitochondria and functionally interact with Cas9 to mediate sequence-specific mtDNA cleavage. Using this novel approach to target the mtDNA, our results provide further evidence that CRISPR-Cas9-mediated gene editing might potentially be used to treat mitochondrial-related diseases.

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGFβ. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant or digenic disorder linked to derepression of the toxic DUX4 gene in muscle. There is currently no pharmacological treatment. The emergence of DUX4 enabled development of cell and animal models that could be used for basic and translational research. Since DUX4 is toxic, animal model development has been challenging, but progress has been made, revealing that tight regulation of DUX4 expression is critical for creating viable animals that develop myopathy. Here, we report such a model - the tamoxifen-inducible FSHD mouse model called TIC-DUX4. Uninduced animals are viable, born in Mendelian ratios, and overtly indistinguishable from WT animals. Induced animals display significant DUX4-dependent myopathic phenotypes at the molecular, histological, and functional levels. To demonstrate the utility of TIC-DUX4 mice for therapeutic development, we tested a gene therapy approach aimed at improving muscle strength in DUX4-expressing muscles using adeno-associated virus serotype 1.Follistatin (AAV1.Follistatin), a natural myostatin antagonist. This strategy was not designed to modulate DUX4 but could offer a mechanism to improve muscle weakness caused by DUX4-induced damage. AAV1.Follistatin significantly increased TIC-DUX4 muscle mass and strength even in the presence of DUX4 expression, suggesting that myostatin inhibition may be a promising approach to treat FSHD-associated weakness. We conclude that TIC-DUX4 mice are a relevant model to study DUX4 toxicity and, importantly, are useful in therapeutic development studies for FSHD.

One strategy for stem cell-based therapy of the cerebral cortex involves the generation and transplantation of functional, histocompatible cortical-like neurons from embryonic stem cells (ESCs). Diploid parthenogenetic Pg-ESCs have recently emerged as a promising source of histocompatible ESC derivatives for organ regeneration but their utility for cerebral cortex therapy is unknown. A major concern with Pg-ESCs is genomic imprinting. In contrast with biparental Bp-ESCs derived from fertilized oocytes, Pg-ESCs harbor two maternal genomes but no sperm-derived genome. Pg-ESCs are therefore expected to have aberrant expression levels of maternally expressed (MEGs) and paternally expressed (PEGs) imprinted genes. Given the roles of imprinted genes in brain development, tissue homeostasis and cancer, their deregulation in Pg-ESCs might be incompatible with therapy. Here, we report that, unexpectedly, only one gene out of 7 MEGs and 12 PEGs was differentially expressed between Pg-ESCs and Bp-ESCs while 13 were differentially expressed between androgenetic Ag-ESCs and Bp-ESCs, indicating that Pg-ESCs but not Ag-ESCs, have a Bp-like imprinting compatible with therapy. In vitro, Pg-ESCs generated cortical-like progenitors and electrophysiologically active glutamatergic neurons that maintained the Bp-like expression levels for most imprinted genes. In vivo, Pg-ESCs participated to the cortical lineage in fetal chimeras. Finally, transplanted Pg-ESC derivatives integrated into the injured adult cortex and sent axonal projections in the host brain. In conclusion, mouse Pg-ESCs generate functional cortical-like neurons with Bp-like imprinting and their derivatives properly integrate into both the embryonic cortex and the injured adult cortex. Collectively, our data support the utility of Pg-ESCs for cortical therapy.

Transferring mouse mutations into specific mouse strain backgrounds can be critical for appropriate analysis of phenotypic effects of targeted genomic alterations and quantitative trait loci. Speed congenic breeding strategies incorporating marker-assisted selection of progeny with the highest percentage target background as breeders for the next generation can produce congenic strains within approximately 5 generations. When mating selected donor males to target strain females, this may require more than 1 year, with each generation lasting 10 to 11 weeks including 3 weeks of gestation and 7 to 8 weeks until the males reach sexual maturity. Because ovulation can be induced in female mice as early as 3 weeks of age, superovulation-aided backcrossing of marker-selected females could accelerate the production of congenic animals by approximately 4 weeks per generation, reducing time and cost. Using this approach, we transferred a transgenic strain of undefined genetic background to >99% C57BL/6J within 10 months, with most generations lasting 7 weeks. This involved less than 60 mice in total, with 9 to 18 animals per generation. Our data demonstrate that high-speed backcrossing through the female germline is feasible and practical with small mouse numbers.

Parent-of-origin imprints have been implicated in the regulation of neural differentiation and brain development. Previously we have shown that, despite the lack of a paternal genome, human parthenogenetic (PG) embryonic stem cells (hESCs) can form proliferating neural stem cells (NSCs) that are capable of differentiation into physiologically functional neurons while maintaining allele-specific expression of imprinted genes. Since biparental ("normal") hESC-derived NSCs (N NSCs) are targeted by immune cells, we characterized the immunogenicity of PG NSCs. Flow cytometry and immunocytochemistry revealed that both N NSCs and PG NSCs exhibited surface expression of human leukocyte antigen (HLA) class I but not HLA-DR molecules. Functional analyses using an in vitro mixed lymphocyte reaction assay resulted in less proliferation of peripheral blood mononuclear cells (PBMC) with PG compared with N NSCs. In addition, natural killer (NK) cells cytolyzed PG less than N NSCs. At a molecular level, expression analyses of immune regulatory factors revealed higher HLA-G levels in PG compared with N NSCs. In line with this finding, MIR152, which represses HLA-G expression, is less transcribed in PG compared with N cells. Blockage of HLA-G receptors ILT2 and KIR2DL4 on natural killer cell leukemia (NKL) cells increased cytolysis of PG NSCs. Together this indicates that PG NSCs have unique immunological properties due to elevated HLA-G expression.

Parthenogenesis is the development of an oocyte without fertilization. Mammalian parthenogenetic (PG) embryos are not viable, but can develop into blastocysts from which embryonic stem cells (ESCs) have been derived in mouse and human. PG ESCs are frequently homozygous for alleles encoding major histocompatibility complex (MHC) molecules. MHC homozygosity permits much more efficient immune matching than MHC heterozygosity found in conventional ESCs, making PG ESCs a promising cell source for cell therapies requiring no or little immune suppression. However, findings of restricted differentiation and proliferation of PG cells in developmental chimeras have cast doubt on the potential of PG ESC derivatives for organ regeneration. To address this uncertainty, we determined whether PG ESC derivatives are effective in rescuing mice with lethal liver failure due to deficiency of fumarylacetoacetate hydrolase (Fah). In developmental chimeras generated by injecting wild-type PG ESCs into Fah-deficient blastocysts, PG ESCs differentiated into hepatocytes that could repopulate the liver, provide normal liver function, and facilitate long-term survival of adult mice. Moreover, after transplantation into adult Fah-deficient mice, PG ESC-derived hepatocytes efficiently engrafted and proliferated, leading to high-level liver repopulation. Our results show that--despite the absence of a paternal genome--PG ESCs can form therapeutically effective hepatocytes.

Tudor domain containing (Tdrd) proteins that are expressed in germ cells are divided into two groups. One group, consisting of TDRD1, TDRKH, TDRD9 and TDRD12, function in piRNA biogenesis and retrotransposon silencing, while the other group including RNF17/TDRD4 and TDRD5-7 are required for spermiogenesis. These Tdrd proteins play distinct roles during male germ cell development. Here, we report the characterization of STK31/TDRD8 in mice. STK31 contains a tudor domain and a serine/threonine kinase domain. We find that STK31 is a cytoplasmic protein in germ cells. STK31 is expressed in embryonic gonocytes of both sexes and postnatal spermatocytes and round spermatids in males. Disruption of the tudor domain and kinase domain of STK31 respectively does not affect fertility in mice. Our data suggest that the function of STK31 may be redundant with other Tdrd proteins in germ cell development.

A micro/nano-fabrication process of a nanochannel electroporation (NEP) array and its application for precise delivery of plasmid for non-viral gene transfection is described. A dip-combing device is optimized to produce DNA nanowires across a microridge array patterned on the polydimethylsiloxane (PDMS) surface with a yield up to 95%. Molecular imprinting based on a low viscosity resin, 1,4-butanediol diacrylate (1,4-BDDA), adopted to convert the microridge-nanowire-microridge array into a microchannel-nanochannel-microchannel (MNM) array. Secondary machining by femtosecond laser ablation is applied to shorten one side of microchannels from 3000 to 50 μm to facilitate cell loading and unloading. The biochip is then sealed in a packaging case with reservoirs and microfluidic channels to enable cell and plasmid loading, and to protect the biochip from leakage and contamination. The package case can be opened for cell unloading after NEP to allow for the follow-up cell culture and analysis. These NEP cases can be placed in a spinning disc and up to ten discs can be piled together for spinning. The resulting centrifugal force can simultaneously manipulate hundreds or thousands of cells into microchannels of NEP arrays within 3 minutes. To demonstrate its application, a 13 kbp OSKM plasmid of induced pluripotent stem cell (iPSC) is injected into mouse embryonic fibroblasts cells (MEFCs). Fluorescence detection of transfected cells within the NEP biochips shows that the delivered dosage is high and much more uniform compared with similar gene transfection carried out by the conventional bulk electroporation (BEP) method.

Uniparental zygotes with two paternal (androgenetic, AG) or two maternal genomes (gynogenetic, GG) cannot develop into viable offsprings but form blastocysts from which pluripotent embryonic stem (ES) cells can be derived. For most organs, it is unclear whether uniparental ES cells can give rise to stably expandable somatic stem cells that can repair injured tissues. Even if previous reports indicated that the capacity of AG ES cells to differentiate in vitro into pan-neural progenitor cells (pNPCs) and into cells expressing neural markers is similar to biparental [normal fertilized (N)] ES cells, their potential for functional neurogenesis is not known. Here we show that murine AG pNPCs give rise to neuron-like cells, which then generate sodium-driven action potentials while maintaining fidelity of imprinted gene expression. Neural engraftment after intracerebral transplantation was achieved only by late (22 days) AG and N pNPCs with in vitro low colony-forming cell (CFC) capacity. However, persisting CFC formation seen, in particular, in early (13 or 16 days) differentiation cultures of N and AG pNPCs correlated with a high incidence of trigerm layer teratomas. As AG ES cells display functional neurogenesis and in vivo stability similar to N ES cells, they represent a unique model system to study the roles of paternal and maternal genomes on neural development and on the development of imprinting-associated brain diseases.

Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.

Histone H1 is an abundant and essential component of chromatin whose precise role in regulating gene expression is poorly understood. Here, we report that a major target of H1-mediated regulation in embryonic stem (ES) cells is the X-linked Rhox homeobox gene cluster. To address the underlying mechanism, we examined the founding member of the Rhox gene cluster-Rhox5-and found that its distal promoter (Pd) loses H1, undergoes demethylation, and is transcriptionally activated in response to loss of H1 genes in ES cells. Demethylation of the Pd is required for its transcriptional induction and we identified a single cytosine in the Pd that, when methylated, is sufficient to inhibit Pd transcription. Methylation of this single cytosine prevents the Pd from binding GA-binding protein (GABP), a transcription factor essential for Pd transcription. Thus, H1 silences Rhox5 transcription by promoting methylation of one of its promoters, a mechanism likely to extend to other H1-regulated Rhox genes, based on analysis of ES cells lacking DNA methyltransferases. The Rhox cluster genes targeted for H1-mediated transcriptional repression are also subject to another DNA methylation-regulated process: Xp imprinting. Remarkably, we found that only H1-regulated Rhox genes are imprinted, not those immune to H1-mediated repression. Together, our results indicate that the Rhox gene cluster is a major target of H1-mediated transcriptional repression in ES cells and that H1 is a candidate to have a role in Xp imprinting.

To be of therapeutic use, autologous stem cells derived from patients with inherited genetic disorders require genetic modification via gene repair or insertion. Here, we present proof of principle that, for diseases associated with dominant alleles (gain-of-function or haploinsufficient loss-of-function), disease allele–free ES cells can be derived from afflicted individuals without genome manipulation. This approach capitalizes on the derivation of uniparental cells, such as parthenogenetic (PG) ES cell lines from disease allele–free gametes. Diploid mammalian uniparental embryos with only maternally (oocyte-) or paternally (sperm-)derived genomes fail early in development due to the nonequivalence of parental genomes caused by genomic imprinting. However, these uniparental embryos develop to the blastocyst stage, allowing the derivation of ES cell lines. Using a mouse model for dominant beta-thalassemia, we developed disease allele–free PG ES cell lines from the oocytes of affected animals. Phenotype correction was obtained in donor-genotype recipients after transplantation of in vitro hematopoietic ES cell derivatives. This genetic correction strategy without gene targeting is potentially applicable to any dominant disease. It could also be the sole approach for larger or more complex mutations that cannot be corrected by homologous recombination.