Fusion of histone variants to Cas9 suppresses non-homologous end joining.

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio by suppressing NHEJ. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 suppressed NHEJ better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.

Large-scale single-cell cloning of genome-edited cultured human cells by On-chip SPiS.

Single-cell cloning is the simplest strategy to isolate genome-edited cell clones, although its scalability has been an issue. Here, we present a protocol to establish genome-edited human cultured cell clones using the On-chip SPiS, a single-cell auto-dispensing device with image recognition technology. Human cultured cells are transfected with plasmids of the CRISPR-Cas9 components, and Cas9-expressing cells are sorted and individually plated into multi-well plates by the On-chip SPiS. For complete details on the use and execution of this protocol, please refer to Takahashi et al. (2022).1.

In Vivo Delivery of Therapeutic Molecules by Transplantation of Genome-Edited Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) have already been used in transplantation therapies. Currently, cells from healthy people are transplanted into patients with diseases. With the rapid evolution of genome editing technology, genetic modification could be applied to enhance the therapeutic effects of iPSCs, such as the introduction of secreted molecules to make the cells a drug delivery system. Here, we addressed this possibility by utilizing a Fabry disease mouse model, as a proof of concept. Fabry disease is caused by the lack of α-galactosidase A (GLA). We previously developed an immunotolerant therapeutic molecule, modified α-N-acetylgalactosaminidase (mNAGA). We confirmed that secreted mNAGA from genome-edited iPSCs compensated for the GLA activity in GLA-deficient cells using an in vitro co-culture system. Moreover, iPSCs transplanted into Fabry model mice secreted mNAGA and supplied GLA activity to the liver. This study demonstrates the great potential of genome-edited iPSCs secreting therapeutic molecules.

Granulin loss of function in human mature brain organoids implicates astrocytes in TDP-43 pathology.

Loss of function (LoF) of TAR-DNA binding protein 43 (TDP-43) and mis-localization, together with TDP-43-positive and hyperphosphorylated inclusions, are found in post-mortem tissue of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those carrying LoF variants in the progranulin gene (GRN). Modeling TDP-43 pathology has been challenging in vivo and in vitro. We present a three-dimensional induced pluripotent stem cell (iPSC)-derived paradigm-mature brain organoids (mbOrg)-composed of cortical-like-astrocytes (iA) and neurons. When devoid of GRN, mbOrgs spontaneously recapitulate TDP-43 mis-localization, hyperphosphorylation, and LoF phenotypes. Mixing and matching genotypes in mbOrgs showed that GRN-/- iA are drivers for TDP-43 pathology. Finally, we rescued TDP-43 LoF by adding exogenous progranulin, demonstrating a link between TDP-43 LoF and progranulin expression. In conclusion, we present an iPSC-derived platform that shows striking features of human TDP-43 proteinopathy and provides a tool for the mechanistic modeling of TDP-43 pathology and patient-tailored therapeutic screening for FTD and ALS.

Genome editing is induced in a binary manner in single human cells.

Even when precise nucleotide manipulations are intended, the outcomes of genome editing can be diverse, often including random insertions and deletions. The combinations and frequencies of these different outcomes in single cells are critical not only in the generation of genetically modified cell lines but also in the evaluation of the clinical effects of genome editing therapies. However, current methods only analyze cell populations, not single cells. Here, we utilized the Single Particle isolation System (SPiS) for the efficient isolation of single cells to systematically analyze genome editing results in individual human cultured cells. As a result, we discovered that genome editing induction has a binary nature, that is, the target alleles of cells tend to be all edited or not edited at all. This study enhances our understanding of the induction pattern of genome editing and provides a new strategy to analyze genome editing outcomes in single cells.

Retinoids rescue ceruloplasmin secretion and alleviate oxidative stress in Wilson's disease-specific hepatocytes.

Wilson's disease (WD) is a copper metabolic disorder caused by a defective ATP7B function. Conventional therapies cause severe side effects and significant variation in efficacy, according to cohort studies. Thus, exploring new therapeutic approaches to prevent progression to liver failure is urgent. To study the physiology and pathology of WD, immortalized cell lines and rodent WD models have been used conventionally; however, a large gap remains among different species as well as in genetic backgrounds among individuals. We generated induced pluripotent stem cells (iPSCs) from four WD patients carrying compound heterozygous mutations in the ATP7B gene. ATP7B loss- and gain-of-functions were further manifested with ATP7B-deficient iPSCs and heterozygously corrected R778L WD patient-derived iPSCs using CRISPR-Cas9-based gene editing. Although the expression of ATP7B protein varied among WD-specific hepatocytes differentiated from these iPSCs, the expression and secretion of ceruloplasmin (Cp), a downstream copper carrier in plasma, were consistently decreased in WD patient-derived and ATP7B-deficient hepatocytes. A transcriptome analysis detected abnormalities in the retinoid signaling pathway and lipid metabolism in WD-specific hepatocytes. Drug screening using WD patient-derived hepatocytes identified retinoids as promising candidates for rescuing Cp secretion. All-trans retinoic acid also alleviates reactive oxygen species production induced by lipid accumulation in WD-specific hepatocytes treated with oleic acid. These patient-derived iPSC-based hepatic models function as effective platforms for the development of potential therapeutics for hepatic steatosis in WD and other fatty liver diseases.

Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies.

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.

Rapid, precise quantification of large DNA excisions and inversions by ddPCR.

The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (> 20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.

Detection and Quantification of HDR and NHEJ Induced by Genome Editing at Endogenous Gene Loci Using Droplet Digital PCR.

Genome editing holds great promise for experimental biology and potential clinical use. To successfully utilize genome editing, it is critical to sensitively detect and quantify its outcomes: homology-directed repair (HDR) and nonhomologous end joining (NHEJ). This has been difficult at endogenous gene loci and instead is frequently done using artificial reporter systems. Here, we describe a droplet digital PCR (ddPCR)-based method to simultaneously measure HDR and NHEJ at endogenous gene loci. This highly sensitive and quantitative method may significantly contribute to a better understanding of DNA repair mechanisms underlying genome editing and to the improvement of genome editing technology by allowing for efficient and systematic testing of many genome editing conditions in parallel.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair.

Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) predominantly induces non-homologous end joining (NHEJ), which generates random insertions or deletions, whereas homology-directed repair (HDR), which generates precise recombination products, is useful for wider applications. However, the factors that determine the ratio of HDR to NHEJ products after CRISPR/Cas9 editing remain unclear, and methods by which the proportion of HDR products can be increased have not yet been fully established. We systematically analyzed the HDR and NHEJ products after genome editing using various modified guide RNAs (gRNAs) and Cas9 variants with an enhanced conformational checkpoint to improve the fidelity at endogenous gene loci in HEK293T cells and HeLa cells. We found that these modified gRNAs and Cas9 variants were able to enhance HDR in both single-nucleotide substitutions and a multi-kb DNA fragment insertion. Our results suggest that the original CRISPR/Cas9 system from the bacterial immune system is not necessarily the best option for the induction of HDR in genome editing and indicate that the modulation of the kinetics of conformational checkpoints of Cas9 can optimize the HDR/NHEJ ratio.

Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity.

Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants.

Detecting Single-Nucleotide Substitutions Induced by Genome Editing.

The detection of genome editing is critical in evaluating genome-editing tools or conditions, but it is not an easy task to detect genome-editing events-especially single-nucleotide substitutions-without a surrogate marker. Here we introduce a procedure that significantly contributes to the advancement of genome-editing technologies. It uses droplet digital polymerase chain reaction (ddPCR) and allele-specific hydrolysis probes to detect single-nucleotide substitutions generated by genome editing (via homology-directed repair, or HDR). HDR events that introduce substitutions using donor DNA are generally infrequent, even with genome-editing tools, and the outcome is only one base pair difference in 3 billion base pairs of the human genome. This task is particularly difficult in induced pluripotent stem (iPS) cells, in which editing events can be very rare. Therefore, the technological advances described here have implications for therapeutic genome editing and experimental approaches to disease modeling with iPS cells.

Using Digital Polymerase Chain Reaction to Detect Single-Nucleotide Substitutions Induced by Genome Editing.

This protocol is designed to detect single-nucleotide substitutions generated by genome editing in a highly sensitive and quantitative manner. It uses a combination of allele-specific hydrolysis probes and a new digital polymerase chain reaction (dPCR) technology called droplet digital PCR (ddPCR). ddPCR partitions a reaction into more than 10,000 nanoliter-scale water-in-oil droplets. As a result, each droplet contains only a few copies of the genome so that ddPCR is able to detect rare genome-editing events without missing them.

Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing.

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs.

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.

Isolation of single-base genome-edited human iPS cells without antibiotic selection.

Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.

Oncostatin M maintains the hematopoietic microenvironment in the bone marrow by modulating adipogenesis and osteogenesis.

The bone marrow (BM) is an essential organ for hematopoiesis in adult, in which proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) is orchestrated by various stromal cells. Alterations of BM hematopoietic environment lead to various hematopoietic disorders as exemplified by the linking of fatty marrow with increased adipogenesis to anemia or pancytopenia. Therefore, the composition of mesenchymal stromal cell (MSC)-derived cells in the BM could be crucial for proper hematopoiesis, but the mechanisms underlying the MSC differentiation for hematopoiesis remain poorly understood. In this study, we show that Oncostatin M (OSM) knock out mice exhibited pancytopenia advancing fatty marrow with age. OSM strongly inhibited adipogenesis from BM MSC in vitro, whereas it enhanced their osteogenesis but suppressed the terminal differentiation. Intriguingly, OSM allowed the MSC-derived cells to support the ex vivo expansion of HSPC effectively as feeder cells. Furthermore, the administration of OSM in lethally irradiated wild-type mice blocked fatty marrow and enhanced the recovery of HSPC number in the BM and peripheral blood cells after engraftment of HSPC. Collectively, OSM plays multiple critical roles in the maintenance and development of the hematopoietic microenvironment in the BM at a steady state as well as after injury.

To divide or not to divide: revisiting liver regeneration.

The liver has a remarkable capacity to regenerate. Even with surgical removal (partial hepatectomy) of 70% of liver mass, the remnant tissue grows to recover the original mass and functions. Liver regeneration after partial hepatectomy has been studied extensively since the 19th century, establishing the long-standing model that hepatocytes, which account for most of the liver weight, proliferate to recover the original mass of the liver. The basis of this model is the fact that almost all hepatocytes undergo S phase, as shown by the incorporation of radioactive nucleotides during liver regeneration. However, DNA replication does not necessarily indicate the execution of cell division, and a possible change in hepatocyte size is not considered in the model. In addition, as 15-30% of hepatocytes in adult liver are binuclear, the difference in nuclear number may affect the mode of cell division during regeneration. Thus, the traditional model seems to be oversimplified. Recently, we developed new techniques to investigate the process of liver regeneration, and revealed interesting features of hepatocytes. In this review, we first provide a historical overview of how the widely accepted model of liver regeneration was established and then discuss some overlooked observations together with our recent findings. Finally, we describe the revised model and perspectives on liver regeneration research.

Hypertrophy and unconventional cell division of hepatocytes underlie liver regeneration.

BACKGROUND: The size of organs and tissues is basically determined by the number and size of their cells. However, little attention has been paid to this fundamental concept. The liver has a remarkable ability to regenerate after surgical resection (partial hepatectomy [PHx]), and hepatocytes account for about 80% of liver weight, so we investigate how the number and size of hepatocytes contribute to liver regeneration in mice. It has been generally accepted that hepatocytes undergo one or two rounds of cell division after 70% PHx. However, ploidy of hepatocytes is known to increase during regeneration, suggesting an unconventional cell cycle. We therefore examine cell cycle of hepatocytes in detail. RESULTS: By developing a method for genetic fate mapping and a high-throughput imaging system of individual hepatocytes, we show that cellular hypertrophy makes the first contribution to liver regeneration; i.e., regeneration after 30% PHx is achieved solely by hypertrophy without cell division, and hypertrophy precedes proliferation after 70% PHx. Proliferation and hypertrophy almost equally contribute to regeneration after 70% PHx. Furthermore, although most hepatocytes enter cell cycle after 70% PHx, not all hepatocytes undergo cell division. In addition, binuclear hepatocytes undergo reductive divisions to generate two mononuclear daughter hepatocytes in some cases. CONCLUSIONS: Our findings demonstrate the importance of hypertrophy and the unconventional cell division cycle of hepatocytes in regeneration, prompting a significant revision of the generally accepted model of liver regeneration.