5'-Modifications improve potency and efficacy of DNA donors for precision genome editing.

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA-repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5'-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.

CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (N4CC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility, exceptional editing accuracy within cells, and high target site density for in vivo genome editing applications.

Rlim-Dependent and -Independent Pathways for X Chromosome Inactivation in Female ESCs.

During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.

Evidence for intermolecular domain exchange in the Fab domains of dimer and oligomers of an IgG1 monoclonal antibody.

Recombinant protein therapeutics have become increasingly useful in combating human diseases, such as cancer and those of genetic origin. One quality concern for protein therapeutics is the content and the structure of the aggregated proteins in the product, due to the potential immunogenicity of these aggregates. Collective efforts have led to a better understanding of some types of protein aggregates, and have revealed the diversity in the structure and cause of protein aggregation. In this work we used a broad range of analytical techniques to characterize the quinary structure (complexes in which each composing unit maintains native quaternary structure) of the stable non-covalent dimer and oligomers of a monoclonal IgG1λ antibody. The results supported a mechanism of intermolecular domain exchange involving the Fab domains of 2 or more IgG molecules. This mechanism can account for the native-like higher order (secondary, tertiary and disulfide bonding) structure, the stability of the non-covalent multimers, and the previously observed partial loss of the antigen-binding sites without changing the antigen-binding affinity and kinetics of the remaining sites (Luo et al., 2009, mAbs 1:491). Furthermore, the previously observed increase in the apparent affinity to various Fcγ receptors (ibid), which may potentially promote immunogenicity, was also explained by the quinary structure proposed here. Several lines of evidence indicated that the formation of multimers by the mechanism of intermolecular domain exchange took place mostly during expression, not in the purified materials. The findings in this work will advance our knowledge of the mechanisms for aggregation in therapeutic monoclonal antibodies.

RLIM is dispensable for X-chromosome inactivation in the mouse embryonic epiblast.

In female mice, two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X chromosome. Later, around implantation, epiblast cells of the inner cell mass that give rise to the embryo reactivate the paternal X chromosome and undergo a random form of XCI (rXCI). Xist, a long non-coding RNA crucial for both forms of XCI, is activated by the ubiquitin ligase RLIM (also known as Rnf12). Although RLIM is required for triggering iXCI in mice, its importance for rXCI has been controversial. Here we show that RLIM levels are downregulated in embryonic cells undergoing rXCI. Using mouse genetics we demonstrate that female cells lacking RLIM from pre-implantation stages onwards show hallmarks of XCI, including Xist clouds and H3K27me3 foci, and have full embryogenic potential. These results provide evidence that RLIM is dispensable for rXCI, indicating that in mice an RLIM-independent mechanism activates Xist in the embryo proper.

Conditional Aurora A deficiency differentially affects early mouse embryo patterning.

Aurora A is a mitotic kinase essential for cell proliferation. In mice, ablation of Aurora A results in mitotic arrest and pre-implantation lethality, preventing studies at later stages of development. Here we report the effects of Aurora A ablation on embryo patterning at early post-implantation stages. Inactivation of Aurora A in the epiblast or visceral endoderm layers of the conceptus leads to apoptosis and inhibition of embryo growth, causing lethality and resorption at approximately E9.5. The effects on embryo patterning, however, depend on the tissue affected by the mutation. Embryos with an epiblast ablation of Aurora A properly establish the anteroposterior axis but fail to progress through gastrulation. In contrast, mutation of Aurora A in the visceral endoderm, leads to posteriorization of the conceptus or failure to elongate the anteroposterior axis. Injection of ES cells into Aurora A epiblast knockout blastocysts reconstitutes embryonic development to E9.5, indicating that the extra-embryonic tissues in these mutant embryos can sustain development to organogenesis stages. Our results reveal new ways to induce apoptosis and to ablate cells in a tissue-specific manner in vivo. Moreover, they show that epiblast-ablated embryos can be used to test the potency of stem cells.

Embryonic stem cell rescue of tremor and ataxia in myelin-deficient shiverer mice.

Transplantation of neural precursor cells has been proposed as a possible approach for replacing missing or damaged central nervous system myelin. Neonatal and adult myelin-deficient shiverer (shi) mice, bearing a mutation of the myelin basic protein (MBP) gene, have been used extensively as hosts for testing cell engraftment, migration, and myelination, but relatively little progress has been made in reversing shi motor deficits. Here we describe a prenatal cell replacement strategy, showing that embryonic stem cells injected into shi blastocyst embryos can generate chimeric mice with strong and widespread immunoreactive MBP expression throughout the brain and a behavioral (motor) phenotype that appears essentially rescued.

Differentiation prevents assessment of neural stem cell pluripotency after blastocyst injection.

Earlier studies reported that neural stem (NS) cells injected into blastocysts appeared to be pluripotent, differentiating into cells of all three germ layers. In this study, we followed in vitro green fluorescent protein (GFP)-labeled NS and embryonic stem (ES) cells injected into blastocysts. Forty-eight hours after injection, significantly fewer blastocysts contained GFP-NS cells than GFP-ES cells. By 96 hours, very few GFP-NS cells remained in blastocysts compared with ES cells. Moreover, 48 hours after injection, GFP-NS cells in blastocysts extended long cellular processes, ceased expressing the NS cell marker nestin, and instead expressed the astrocytic marker glial fibrillary acidic protein. GFP-ES cells in blastocysts remained morphologically undifferentiated, continuing to express the pluripotent marker stage-specific embryonic antigen-1. Selecting cells from the NS cell population that preferentially formed neurospheres for injection into blastocysts resulted in identical results. Consistent with this in vitro behavior, none of almost 80 mice resulting from NS cell-injected blastocysts replaced into recipient mothers were chimeric. These results strongly support the idea that NS cells cannot participate in chimera formation because of their rapid differentiation into glia-like cells. Thus, these results raise doubts concerning the pluripotency properties of NS cells.