Mouse genome rewriting and tailoring of three important disease loci
Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases1,2. Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe 'mammalian switching antibiotic resistance markers progressively for integration' (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing.
A conditional counterselectable Piga knockout in mouse embryonic stem cells for advanced genome writing applications
Overwriting counterselectable markers is an efficient strategy for removing wild-type DNA or replacing it with payload DNA of interest. Currently, one bottleneck of efficient genome engineering in mammals is the shortage of counterselectable (negative selection) markers that work robustly without affecting organismal developmental potential. Here, we report a conditional Piga knockout strategy that enables efficient proaerolysin-based counterselection in mouse embryonic stem cells. The conditional Piga knockout cells show similar proaerolysin resistance as full (non-conditional) Piga deletion cells, which enables the use of a PIGA transgene as a counterselectable marker for genome engineering purposes. Native Piga function is readily restored in conditional Piga knockout cells to facilitate subsequent mouse development. We also demonstrate the generality of our strategy by engineering a conditional knockout of endogenous Hprt. Taken together, our work provides a new tool for advanced mouse genome writing and mouse model establishment.
Klf5 establishes bi-potential cell fate by dual regulation of ICM and TE specification genes
Early blastomeres of mouse preimplantation embryos exhibit bi-potential cell fate, capable of generating both embryonic and extra-embryonic lineages in blastocysts. Here we identify three major two-cell-stage (2C)-specific endogenous retroviruses (ERVs) as the molecular hallmark of this bi-potential plasticity. Using the long terminal repeats (LTRs) of all three 2C-specific ERVs, we identify KrÃ¼ppel-like factor 5 (Klf5) as their major upstream regulator. Klf5 is essential for bi-potential cell fate; a single Klf5-overexpressing embryonic stem cell (ESC) generates terminally differentiated embryonic and extra-embryonic lineages in chimeric embryos, and Klf5 directly induces inner cell mass (ICM) and trophectoderm (TE) specification genes. Intriguingly, Klf5 and Klf4 act redundantly during ICM specification, whereas Klf5 deficiency alone impairs TE specification. Klf5 is regulated by multiple 2C-specific transcription factors, particularly Dux, and the Dux/Klf5 axis is evolutionarily conserved. The 2C-specific transcription program converges on Klf5 to establish bi-potential cell fate, enabling a cell state with dual activation of ICM and TE genes.
Analysis of urgent/emergent conversions from monitored anesthesia care to general anesthesia with airway instrumentation
BACKGROUND:Monitored Anesthesia Care (MAC) is an anesthetic service involving the titration of sedatives/analgesics to achieve varying levels of sedation while avoiding general anesthesia (GA) and airway instrumentation. The goal of our study was to determine the overall incidence of conversion from MAC to general anesthesia with airway instrumentation and elucidate reasons and risk factors for conversion. METHODS:In this retrospective observational study, all non-obstetric adult patients who received MAC from July 2002 to July 2015 at Mount Sinai Hospital were electronically screened for inclusion via a clinical database. Patient, procedure, anesthetic, and practitioner data were all collected and analyzed to generate descriptive analyses. Subsequent univariate and multivariate analyses were used to identify specific risk factors associated with conversion to GA. RESULTS:Overall, 0.50% (1097/219,061) of MAC cases were converted to GA. Approximately half of conversions were due to the patient's "intolerance" of MAC (with or without failed regional anesthesia), while the other half were due to physiologic derangements. Body mass index, male sex, American Society of Anesthesiologists Physical Status Classification, anesthesia team composition, and surgical specialty were all associated with risk of conversion to GA. CONCLUSIONS:This is one of the first and largest retrospective studies aimed at identifying reasons and risk factors associated with the conversion of MAC to GA. These findings may be used to help better anticipate or prevent these events.
Genetic variation of staphylococcal LukAB toxin determines receptor tropism
Staphylococcus aureus has evolved into diverse lineages, known as clonal complexes (CCs), which exhibit differences in the coding sequences of core virulence factors. Whether these alterations affect functionality is poorly understood. Here, we studied the highly polymorphic pore-forming toxin LukAB. We discovered that the LukAB toxin variants produced by S. aureus CC30 and CC45 kill human phagocytes regardless of whether CD11b, the previously established LukAB receptor, is present, and instead target the human hydrogen voltage-gated channel 1 (HVCN1). Biochemical studies identified the domain within human HVCN1 that drives LukAB species specificity, enabling the generation of humanized HVCN1 mice with enhanced susceptibility to CC30 LukAB and to bloodstream infection caused by CC30 S. aureus strains. Together, this work advances our understanding of an important S. aureus toxin and underscores the importance of considering genetic variation in characterizing virulence factors and understanding the tug of war between pathogens and the host.
Context-Dependent Requirement of Euchromatic Histone Methyltransferase Activity during Reprogramming to Pluripotency
Methylation of histone 3 at lysine 9 (H3K9) constitutes a roadblock for cellular reprogramming. Interference with methyltransferases or activation of demethylases by the cofactor ascorbic acid (AA) facilitates the derivation of induced pluripotent stem cells (iPSCs), but possible interactions between specific methyltransferases and AA treatment remain insufficiently explored. We show that chemical inhibition of the methyltransferases EHMT1 and EHMT2 counteracts iPSC formation in an enhanced reprogramming system in the presence of AA, an effect that is dependent on EHMT1. EHMT inhibition during enhanced reprogramming is associated with rapid loss of H3K9 dimethylation, inefficient downregulation of somatic genes, and failed mesenchymal-to-epithelial transition. Furthermore, transient EHMT inhibition during reprogramming yields iPSCs that fail to efficiently give rise to viable mice upon blastocyst injection. Our observations establish novel functions of H3K9 methyltransferases and suggest that a functional balance between AA-stimulated enzymes and EHMTs supports efficient and less error-prone iPSC reprogramming to pluripotency.
Physiological expression and function of the MDR1 transporter in cytotoxic T lymphocytes
Multidrug resistance-1 (MDR1) acts as a chemotherapeutic drug efflux pump in tumor cells, although its physiological functions remain enigmatic. Using a recently developed MDR1-knockin reporter allele (Abcb1aAME), we found that constitutive MDR1 expression among hematopoietic cells was observed in cytolytic lymphocytes-including CD8+ cytotoxic T lymphocytes (CTLs) and natural killer cells-and regulated by Runt-related (Runx) transcription factors. Whereas MDR1 was dispensable for naive CD8+ T cell development, it was required for both the normal accumulation of effector CTLs following acute viral infection and the protective function of memory CTLs following challenge with an intracellular bacterium. MDR1 acted early after naive CD8+ T cell activation to suppress oxidative stress, enforce survival, and safeguard mitochondrial function in nascent CTLs. These data highlight an important endogenous function of MDR1 in cell-mediated immune responses and suggest that ongoing efforts to intentionally inhibit MDR1 in cancer patients could be counterproductive.
The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids
Exploiting species specificity to understand the tropism of a human-specific toxin
Many pathogens produce virulence factors that are specific toward their natural host. Clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) isolates are highly adapted to humans and produce an array of human-specific virulence factors. One such factor is LukAB, a recently identified pore-forming toxin that targets human phagocytes by binding to the integrin component CD11b. LukAB exhibits strong tropism toward human, but not murine, CD11b. Here, phylogenetics and biochemical studies lead to the identification of an 11-residue domain required for the specificity of LukAB toward human CD11b, which is sufficient to render murine CD11b compatible with toxin binding. CRISPR-mediated gene editing was used to replace this domain, resulting in a "humanized" mouse. In vivo studies revealed that the humanized mice exhibit enhanced susceptibility to MRSA bloodstream infection, a phenotype mediated by LukAB. Thus, these studies establish LukAB as an important toxin for MRSA bacteremia and describe a new mouse model to study MRSA pathobiology.
Widespread Transcriptional Scanning in the Testis Modulates Gene Evolution Rates
The testis expresses the largest number of genesÂ ofÂ any mammalian organ, a finding that has long puzzled molecular biologists. Our single-cell transcriptomic data of human and mouse spermatogenesis provide evidence that this widespread transcription maintains DNA sequence integrity in the male germline by correcting DNA damage through a mechanism we term transcriptional scanning. We find that genes expressed during spermatogenesis display lower mutation rates on the transcribed strand and have low diversity in the population. Moreover, this effect is fine-tuned by the level of gene expression during spermatogenesis. The unexpressed genes, which in our model do not benefit from transcriptional scanning, diverge faster over evolutionary timescales and are enriched for sensory and immune-defense functions. Collectively, we propose that transcriptional scanning shapes germline mutation signatures and modulates mutation rates in a gene-specific manner, maintaining DNA sequence integrity for the bulk of genes but allowing for faster evolution in a specific subset.