Biology subjects

Lee, K.

Publications and source records attributed to Lee, K..

At least 163 records · Page 9Linked to original sources

bioRxiv2023

Cell-type-specific inhibitory circuitry from a connectomic census of mouse visual cortex

Mammalian cortex features a vast diversity of neuronal cell types, each with characteristic anatomical, molecular and functional properties. Synaptic connectivity powerfully shapes how each cell type participates in the cortical circuit, but mapping connectivity rules at the resolution of distinct cell types remains difficult. Here, we used millimeter-scale volumetric electron microscopy1 to investigate the connectivity of all inhibitory neurons across a densely-segmented neuronal population of 1352 cells spanning all layers of mouse visual cortex, producing a wiring diagram of inhibitory connections with more than 70,000 synapses. Taking a data-driven approach inspired by classical neuroanatomy, we classified inhibitory neurons based on the relative targeting of dendritic compartments and other inhibitory cells and developed a novel classification of excitatory neurons based on the morphological and synaptic input properties. The synaptic connectivity between inhibitory cells revealed a novel class of disinhibitory specialist targeting basket cells, in addition to familiar subclasses. Analysis of the inhibitory connectivity onto excitatory neurons found widespread specificity, with many interneurons exhibiting differential targeting of certain subpopulations spatially intermingled with other potential targets. Inhibitory targeting was organized into "motif groups," diverse sets of cells that collectively target both perisomatic and dendritic compartments of the same excitatory targets. Collectively, our analysis identified new organizing principles for cortical inhibition and will serve as a foundation for linking modern multimodal neuronal atlases with the cortical wiring diagram.

neuroscience↗

bioRxiv2023

Divergent Proteome Reactivity Influences Arm-Selective Activation of Pharmacological Endoplasmic Reticulum Proteostasis Regulators

Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the Unfolded Protein Response (UPR) has proven useful for ameliorating proteostasis deficiencies in a variety of etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino-p-cresol substructure affording a quinone methide, which then covalently modifies a subset of ER protein disulfide isomerases (PDIs). Intriguingly, another compound identified in this screen, AA132, also contains a 2-amino-p-cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent PDI modification. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. Paradoxically, activated AA132 reacts slower with PDIs, indicating it is less reactive than activated AA147. This suggests that the higher labeling of PDIs observed with activated AA132 can be attributed to its lower reactivity, which allows this activated compound to persist longer in the cellular environment prior to quenching by endogenous nucleophiles. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and allows for selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

biochemistry↗

bioRxiv2023

Listeria monocytogenes requires DHNA-dependent intracellular redox homeostasis facilitated by Ndh2 for survival and virulence

Listeria monocytogenes is a remarkably well-adapted facultative intracellular pathogen that can thrive in a wide range of ecological niches. L. monocytogenes maximizes its ability to generate energy from diverse carbon sources using a respiro-fermentative metabolism that can function under both aerobic and anaerobic conditions. Cellular respiration maintains redox homeostasis by regenerating NAD+ while also generating a proton motive force (PMF). The end products of the menaquinone (MK) biosynthesis pathway are essential to drive both aerobic and anaerobic cellular respiration. We previously demonstrated that intermediates in the MK biosynthesis pathway, notably 1,4-dihydroxy-2-naphthoate (DHNA), are required for the survival and virulence of L. monocytogenes independent of their role in respiration. Furthermore, we found that restoration of NAD+/NADH ratio through expression of water-forming NADH oxidase (NOX) could rescue phenotypes associated with DHNA deficiency. Here we extend these findings to demonstrate that endogenous production or direct supplementation of DHNA restored both the cellular redox homeostasis and metabolic output of fermentation in L. monocytogenes. Further, exogenous supplementation of DHNA rescues the in vitro growth and ex vivo virulence of L. monocytogenes DHNA-deficient mutants. Finally, we demonstrate that exogenous DHNA restores redox balance in L. monocytogenes specifically through the recently annotated NADH dehydrogenase Ndh2, independent of the extracellular electron transport (EET) pathway. These data suggest that the production of DHNA may represent an additional layer of metabolic adaptability by L. monocytogenes to drive energy metabolism in the absence of respiration-favorable conditions.

microbiology↗

bioRxiv2022

Haploid-resolved and chromosome-scale genome assembly in hexa-autoploid sweetpotato (Ipomoea batatas (L.) Lam)

Sweetpotato (Ipomoea batatas (L.) Lam) is the worlds seventh most important food crop by production quantity. Cultivated sweetpotato is a hexaploid (2n = 6x = 90), and its genome (B1B1B2B2B2B2) is quite complex due to polyploidy, self-incompatibility, and high heterozygosity. Here we established a haploid-resolved and chromosome-scale de novo assembly of autohexaploid sweetpotato genome sequences. Before constructing the genome, we created chromosome-scale genome sequences in I. trifida using a highly homozygous accession, Mx23Hm, with PacBio RSII and Hi-C reads. Haploid-resolved genome assembly was performed for a sweetpotato cultivar, Xushu18 by hybrid assembly with Illumina paired-end (PE) and mate-pair (MP) reads, 10X genomics reads, and PacBio RSII reads. Then, 90 chromosome-scale pseudomolecules were generated by aligning the scaffolds onto a sweetpotato linkage map. De novo assemblies were also performed for chloroplast and mitochondrial genomes in I. trifida and sweetpotato. In total, 34,386 and 175,633 genes were identified on the assembled nucleic genomes of I. trifida and sweetpotato, respectively. Functional gene annotation and RNA-Seq analysis revealed locations of starch, anthocyanin, and carotenoid pathway genes on the sweetpotato genome. This is the first report of chromosome-scale de novo assembly of the sweetpotato genome. The results are expected to contribute to genomic and genetic analyses of sweetpotato.

genomics↗

bioRxiv2022

Large-scale unsupervised discovery of excitatory morphological cell types in mouse visual cortex

Neurons in the neocortex exhibit astonishing morphological diversity which is critical for properly wiring neural circuits and giving neurons their functional properties. However, the organizational principles underlying this morphological diversity remain an open question. Here, we took a data-driven approach using graph-based machine learning methods to obtain a low-dimensional morphological "bar code" describing more than 30,000 excitatory neurons in mouse visual areas V1, AL and RL that were reconstructed from the millimeter scale MICrONS serial-section electron microscopy volume. Contrary to previous classifications into discrete morphological types (m-types), our data-driven approach suggests that the morphological landscape of cortical excitatory neurons is better described as a continuum, with a few notable exceptions in layers 5 and 6. Dendritic morphologies in layers 2-3 exhibited a trend towards a decreasing width of the dendritic arbor and a smaller tuft with increasing cortical depth. Inter-area differences were most evident in layer 4, where V1 contained more atufted neurons than higher visual areas. Moreover, we discovered neurons in V1 on the border to layer 5 which avoided deeper layers with their dendrites. In summary, we suggest that excitatory neurons morphological diversity is better understood by considering axes of variation than using distinct m-types.

neuroscience↗

bioRxiv2022

Tools for comprehensive reconstruction and analysis of Drosophila motor circuits

Like the vertebrate spinal cord, the insect ventral nerve cord (VNC) mediates limb sensation and motor control. Here, we apply automated tools for electron microscopy volume alignment, neuron segmentation, and synapse prediction toward creating a connectome of an adult female Drosophila VNC. To interpret a connectome, it is crucial to know its relationship with the rest of the body. We therefore mapped the muscle targets of leg and wing motor neurons in the connectome by comparing their morphology to genetic driver lines, dye fills, and X-ray nano-tomography of the fly leg and wing. Knowing the outputs of the connectome allowed us to identify neural circuits that coordinate the wings and legs during escape takeoff. We provide the reconstruction of VNC circuits and motor neuron atlas, along with tools for programmatic and interactive access, as community resources to support experimental and theoretical studies of how the fly nervous system controls behavior.

neuroscience↗

bioRxiv2022

Leptospermum extract (QV0) suppresses pleural mesothelioma tumour growth in vitro and in vivo by mitochondrial dysfunction associated apoptosis

Pleural mesothelioma (PM) is a highly aggressive, fast-growing asbestos-induced cancer with limited effective treatments. There has been an interest in using naturally occurring anticancer agents derived from plant materials for the treatment of PM. However, it is unclear if aqueous extract from the Leptospermum polygalifolium (QV0) has activity against PM. Here we investigated the anti-cancer property of QV0 in vitro and in vivo. Animals treated with Defender(R) (QV0 dietary supply) exhibited a reduced tumour size over 30 days, which was associated with an average extended of seven days mouse life. There was no liver toxicity, nor increased blood glucose post-treatment in animals treated with Defender(R). Moreover, QV0 suppressed the growth of 13 cancer cell lines in a dose-dependent manner, effective at concentrations as low as 0.02% w/v. This response was found to be associated with inhibited cell migration, proliferation, and colony formation, but without evident cell cycle alteration. We observed mitochondrial dysfunction post QV0 treatment, as evidenced by significantly decreased basal and maximal oxygen consumption rates. Significantly enhanced tumour apoptosis was observed in the Defender(R)-treated animals, correlating with mitochondrial dysfunction. To the best of our knowledge, this study constitutes the first demonstration of an improved host survival (without adverse effects) response in a QV0-treated PM mouse model, associated with an evident inhibition of PM cell growth and mitochondrial dysfunction-related enhancement of tumour apoptosis. ImportanceA major problem with cancer chemotherapy or immunotherapy is the severe adverse effects associated with normal tissue damage. PM is known to be treatment resistant and has poor a prognosis, therefore new therapeutic treatment options are urgently needed. In the present study, we explored the potential utility of a Leptospermum extract (QV0) as a treatment option for mesothelioma. We demonstrated for the first time that QV0 exhibits an anti-tumour response in mesothelioma, without any associated adverse effects observed in the PM mouse model. These findings provide a rationale for early-stage clinical trials. We anticipate that prospective translational research will lead to the clinical implementation of a novel QV0-based treatment strategy that will ultimately benefit PM patients.

cancer biology↗

bioRxiv2022

CD244 regulates both innate and adaptive immune axes in melanoma by inhibiting autophagy-mediated M1 macrophage maturation

Accumulating data have highlighted the role of monocytes/macrophages in immune escape by generating immunologically "cold" tumors that do not respond to immunotherapy. CD244 (SLAMF4, 2B4), a member of the signaling lymphocyte activation molecule family, is expressed on myeloid cells, but its precise role has not been elucidated. Using monocyte lineage-specific CD244-deficient (LysM-cre+/-CD244fl/fl;cKO) mice challenged with B16F10 melanoma, we report for the first time that CD244 negatively regulates tumor immunity by inhibiting the differentiation and functional maturation of CD11b+Ly6ChiF4/80lo monocytes into CD11b+Ly6CloF4/80hi macrophages within the tumor microenvironment. CD244-deficient macrophages more effectively activated antigen-specific T cell responses compared to WT macrophages, thus delaying tumor growth in the B16F10 melanoma model. Moreover, combinatorial intervention of anti-PD-L1 antibodies with CD244-KO BMDM markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with WT BMDM. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset (SCP398 from single-cell portal), revealed 221 differentially expressed genes of CD244- monocytes/macrophages were associated with phagocytosis, antigen presentation, and autophagy. Additionally, cell type deconvolution analysis within melanoma patients bulk RNA-seq datasets from TCGA database, revealed presence of CD244- monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. Hence, we proposed that CD244 serve as a critical immune checkpoint receptor on macrophages, and CD244-deficient macrophages may represent a novel therapeutic modality to convert immunologically "cold" tumors to "hot" tumors, which can function synergistically with checkpoint blockade therapies.

immunology↗

bioRxiv2022

On the role and regulation of glycogen catabolic isoenzymes in Synechocystis sp. PCC6803

Glycogen serves as the main carbon storage polymer in many organisms and is widespread across all domains of life. In cyanobacteria, glycogen degradation is crucial for metabolic transitions during dark phases or during resuscitation from nitrogen starvation. Like many other cyanobacteria, Synechocystis sp. PCC 6803 possesses multiple homologues of glycogen catabolizing enzymes, though their specific roles and regulatory mechanisms are only partially understood. Here we demonstrate through biochemical analysis that the glycogen phosphorylase GlgP1, known to promote high temperature acclimation, is uniquely regulated by a C-terminal redox switch found certain cyanobacteria. This is the first evidence of redox regulation of a prokaryotic glycogen degrading enzyme. Notably, GlgP1 is activated via oxidation by reactive oxygen species and inactivated by reducing agents, with thioredoxin being the most effective inhibitor tested. The physiological implications of this redox regulation are discussed. Additionally, a biochemically characterization of the two glycogen debranching isoenzymes GlgX1 and GlgX2 revealed that only GlgX1 exhibits debranching activity, while GlgX2 does not. Mutant analysis confirmed that GlgX1 plays an essential role in glycogen mobilization, being crucial for resuscitation from chlorosis and survival during extended dark periods. In contrast the physiological function of GlgX2 remains unclear.

microbiology↗

bioRxiv2022

Survivin is a mechanosensitive cell cycle regulator in vascular smooth muscle cells

Stiffened arteries are a pathology of atherosclerosis, hypertension, and coronary artery disease and a key risk factor for cardiovascular disease events. The increased stiffness of arteries triggers the hypermigration and hyperproliferation of vascular smooth muscle cells (VSMCs), leading to neointimal hyperplasia and accelerated neointima formation, but the mechanism of this trigger is not known. Our analyses of whole-transcriptome microarray data sets from mouse VSMCs cultured on stiff hydrogels simulating arterial pathology and from injured mouse femoral arteries revealed 80 genes that were differentially regulated (74 upregulated and 6 downregulated) relative to expression in control VSMCs cultured on soft hydrogels and in uninjured femoral arteries. A functional enrichment analysis revealed that these stiffness-sensitive genes are linked to cell cycle progression and proliferation. Furthermore, we found that survivin, a member of the inhibitor of apoptosis protein family, mediates stiffness-sensitive cell cycling and proliferation in vivo and in vitro as determined by gene network and pathway analyses, RT-qPCR, and immunoblotting. The stiffness signal is mechanotransduced via FAK and Rac signaling to regulate survivin expression, establishing a regulatory pathway for how the stiffness of the cellular microenvironment affects VSMC behaviors. Our findings indicate that survivin is necessary for VSMC cycling and proliferation and regulates stiffness-responsive phenotypes.

cell biology↗

bioRxiv2022

Flexible, Scalable, High Channel Count Stereo-Electrode for Recording in the Human Brain

Over the past decade, stereotactically placed electrodes have become the gold standard for deep brain recording and stimulation for a wide variety of neurological and psychiatric diseases. Current electrodes, however, are limited in their spatial resolution and ability to record from small populations of neurons, let alone individual neurons. Here, we report on a novel, reconfigurable, monolithically integrated human-grade flexible depth electrode capable of recording from up to 128 channels and able to record at a depth of 10 cm in brain tissue. This thin, stylet-guided depth electrode is capable of recording local field potentials and single unit neuronal activity (action potentials), validated across species. This device represents a major new advance in manufacturing and design approaches which extends the capabilities of a mainstay technology in clinical neurology. One-Sentence SummaryA human-grade thin-film depth electrode offers new opportunities in spatial and temporal resolution for recording brain activity.

neuroscience↗

bioRxiv2022

Survivin Regulates Intracellular Stiffness and Extracellular Matrix Production in Vascular Smooth Muscle Cells

Vascular dysfunction is a common cause of cardiovascular diseases characterized by the narrowing and stiffening of arteries, such as atherosclerosis, restenosis, and hypertension. Arterial narrowing results from the aberrant proliferation of vascular smooth muscle cells (VSMCs) and their increased synthesis and deposition of extracellular matrix (ECM) proteins. These, in turn, are modulated by arterial stiffness, but the mechanism for this is not fully understood. We found that survivin (an inhibitor of apoptosis) is an important regulator of stiffness-mediated ECM synthesis and intracellular stiffness in VSMCs. Whole-transcriptome analysis and cell culture experiments showed that survivin expression is upregulated in injured femoral arteries in mice and in human VSMCs cultured on stiff fibronectin-coated hydrogels. Suppressed expression of survivin in human VSMCs and mouse embryonic fibroblasts decreased the stiffness-mediated expression of ECM components implicated in arterial stiffness, namely, collagen-I, fibronectin, and lysyl oxidase. By contrast, expression of these proteins was upregulated by the overexpression of survivin in human VSMCs cultured on soft hydrogels. Atomic force microscopy analysis showed that suppressed or enhanced expression of survivin decreases or increases intracellular stiffness, respectively. These findings suggest a novel mechanism by which survivin modulates arterial stiffness.

cell biology↗

bioRxiv2022

Disease-causing variant recommendation system for clinical genome interpretation with adjusted scores for artefactual variants

BackgroundIn the process of finding the causative variant of rare diseases (RD), accurate assessment and prioritization of genetic variants is essential. Although quality control (QC) of genetic variants is strictly performed, the presence of artefactual variants in the remaining set of variants can deteriorate the process. Variant QC and prioritization have been treated as separate processes, leading to limited efficiency and risk of misdiagnosis. ResultsWe developed a disease-causing variant recommendation system that integrates quality control into variant prioritization by adjusting scores for artefactual variants. We confirmed that the QC-related features of the variants contribute to a significant performance improvement. For genomic data from 2,878 patients with rare disorders, the recall rate of finding causative variants was 0.961 for the top 5 ranked variants. We also found that our system recognized the anomaly of QC-related features, so that the scores of artifactual variants to be disease-causing were assessed relatively low. ConclusionsIntegration of variant QC and prioritization help reduce the risk of misdiagnosis based on artefactual variants and increase the effectiveness of clinical genome interpretation.

genetics↗

bioRxiv2022

mTOR inhibitor-independent Autophagy Activator Ameliorates Cellular Tauopathy and Prionopathy Neurodegeneration Phenotypes

Autophagy-lysosomal impairment is an early and prominent feature of neurodegeneration. Autophagy activation reduces protein aggregates and lipid level abnormalities. We performed a high-content imaging-based screen assessing 940,000 small molecules to identify those that reduce lipid droplet numbers. Of 77 validated, structurally diverse hits, 24 increased autophagy flux reporter activity, consistent with accelerated lipid droplet clearance by lipophagy. Of these, we show that CCT020312 activates autophagy independently of mammalian target of rapamycin (mTOR) inhibition, to avoid immunosuppression. CCT020312 reduced insoluble phosphorylated tau levels and tau-mediated neuronal stress vulnerability, as well as reducing intracellular A{beta} levels within directly induced neurons bearing epigenetic marks of aging derived from Alzheimers patient fibroblasts. Moreover, CCT020312 cleared mutant prion protein aggregates and normalized trafficking deficiencies in axons of a cellular model of familial prion disease. Autophagy is widely considered a promising strategy to attenuate neurodegeneration, and here we introduce a strategy to discover new pharmacology.

cell biology↗

bioRxiv2022

Physiological constraints on the rapid dopaminergic modulation of striatal reward activity

While the contribution of dopaminergic (DA) neurons to associative learning is firmly established, their importance for influencing imminent behavior on short (subsecond) timescales is less clear. Mechanistically, it is thought that DA neurons drive these behavioral changes because of their ability to rapidly alter striatal spiking activity. However, due to limitations of previous approaches, the straightforward prediction that striatal spiking is rapidly influenced by physiologically relevant DA signals has not been rigorously tested. Here, we monitored changes in spiking responses in the ventral striatum while transiently reducing or increasing DA levels. Contrary to the predicted effect, neither spontaneous nor reward-evoked striatal spiking activity was strongly influenced by optogenetic manipulations, except when DA exceeded reward-matched levels. These findings challenge the view that DA plays a major role in rapidly influencing striatal activity. Finally, they suggest a need to distinguish between the modulatory functions of DA under physiological and supra-physiological conditions.

neuroscience↗

bioRxiv2022

Single cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation.

Stem cell technologies provide new opportunities for modeling cells in the healthy and diseased states and for regenerative medicine. In both cases developmental knowledge as well as the quality and molecular properties of the cells are essential for their future application. In this study we identify developmental factors important for the differentiation of human embryonic stem cells (hESCs) into midbrain dopaminergic (mDA) neurons. We found that Laminin-511, and dual canonical and non-canonical WNT activation followed by GSK3{beta} inhibition plus FGF8b, improved midbrain patterning. In addition, mDA neurogenesis and differentiation was enhanced by activation of liver X receptors and inhibition of fibroblast growth factor signaling. Moreover, single-cell RNA-sequencing analysis revealed a developmental dynamics similar to that of the endogenous human ventral midbrain and the emergence of high quality molecularly-defined midbrain cell types, including mDA neurons that become functional. Thus, our study identifies novel factors important for human midbrain development and opens the door for a future application of molecularly-defined hESC-derived midbrain cell types in Parkinsons disease.

neuroscience↗

bioRxiv2022

RUNX1-deficient human megakaryocytes demonstrate thrombopoietic and platelet half-life and functional defects in an in vivo murine model: Therapeutic implications

Heterozygous defects in runt-related transcription factor-1 (RUNX1) are causative of a familial platelet disorder with associated myeloid malignancy (FPDMM). Since RUNX1-deficient animal models do not mimic FPDMMs bleeding disorder or leukemic risk, establishment of a proper model system is critical to understand the underlying mechanisms of the observed phenotype and to identify therapeutic interventions. We previously reported an in vitro-megakaryopoiesis system using human CD34+-hematopoietic stem and progenitor cells that recapitulated the FPDMM quantitative megakaryocyte defect by decreasing RUNX1 expression using a lentiviral short-hairpin RNA (shRNA for RUNX1 or shRX) strategy. We now show that shRX-megakaryocytes have a marked reduction in agonist responsiveness. We then infused shRX-megakaryocytes into immunocompromised NOD-SCID gamma (NSG) mice and demonstrated that these megakaryocytes released fewer platelets than megakaryocytes transfected with a non-targeting shRNA, and these platelets had a diminished half-life. The platelets were also poorly responsive to agonists, unable to correct thrombus formation in NSG mice homozygous for a R1326H mutation in von Willebrand Factor (VWFR1326H), which switches species-binding specificity of the VWF from mouse to human glycoprotein Ib. A small-molecule inhibitor RepSox, which blocks the transforming-growth factor beta pathway, and which rescued defective megakaryopoiesis in vitro, corrected the thrombopoietic defect, platelet half-life and agonist response, and thrombus formation in NSG/VWFR1326H mice. Thus, this model recapitulates the defect in FPDMM megakaryocytes and platelets, identifies previously unrecognized defects in thrombopoiesis and platelet half-life, and demonstrates, for the first time, reversal of RUNX1 deficiencys hemostatic defects by a drug. Key PointsO_LIRUNX1-deficient megakaryocytes exhibit thrombopoietic and platelet defects in NSG/VWFR1326H mice. C_LIO_LIPre-exposure of RUNX1-deficient megakaryocytes to a TGF{beta}1-pathway inhibitor ameliorated both defects, correcting hemostasis. C_LI

cancer biology↗

bioRxiv2022

Quantitative comparison of CRISPR-Cas9-mediated mutation efficiency between mice and MEFs using digital PCR assays

The creation of mutant mice has been invaluable for advancing biomedical science, but is too time- and resource-intensive for investigating the full range of mutations and polymorphisms. Cell culture models are therefore an invaluable complement to mouse models, especially for cell-autonomous pathways like the circadian clock. In this study, we quantitatively assessed the use of CRISPR to create cell models in MEFs as compared to mouse models. We generated two point mutations in the clock genes Per1 and Per2 in mice and in MEFs using the same sgRNAs and repair templates for HDR and quantified the frequency of the mutations by digital PCR. The frequency was about an order of magnitude higher in mouse zygotes compared to that in MEFs. However, the mutation frequency in MEFs was still high enough for clonal isolation by simple screening of a few dozen individual cells. The Per mutant cells that we generated provide important new insights into the role of the PAS domain in regulating PER phosphorylation, a key aspect of the circadian clock mechanism. Accurate quantification of the mutation frequency in bulk MEF populations provides a critical basis for optimizing CRISPR protocols and time/resource planning for generating cell models for further studies.

neuroscience↗