Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.

Distinct fibroblast subsets regulate lacteal integrity through YAP/TAZ-induced VEGF-C in intestinal villi

Emerging evidence suggests that intestinal stromal cells (IntSCs) play essential roles in maintaining intestinal homeostasis. However, the extent of heterogeneity within the villi stromal compartment and how IntSCs regulate the structure and function of specialized intestinal lymphatic capillary called lacteal remain elusive. Here we show that selective hyperactivation or depletion of YAP/TAZ in PDGFR beta(+) IntSCs leads to lacteal sprouting or regression with junctional disintegration and impaired dietary fat uptake. Indeed, mechanical or osmotic stress regulates IntSC secretion of VEGF-C mediated by YAP/TAZ. Single-cell RNA sequencing delineated novel subtypes of villi fibroblasts that upregulate Vegfc upon YAP/TAZ activation. These populations of fibroblasts were distributed in proximity to lacteal, suggesting that they constitute a peri-lacteal microenvironment. Our findings demonstrate the heterogeneity of IntSCs and reveal that distinct subsets of villi fibroblasts regulate lacteal integrity through YAP/TAZ-induced VEGF-C secretion, providing new insights into the dynamic regulatory mechanisms behind lymphangiogenesis and lymphatic remodeling.Peer reviewe

In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration.

Induction of Neuronal Death by Microglial AGE-Albumin: Implications for Alzheimer’s Disease

Advanced glycation end products (AGEs) have long been considered as potent molecules promoting neuronal cell death and contributing to neurodegenerative disorders such as Alzheimer’s disease (AD). In this study, we demonstrate that AGE-albumin, the most abundant AGE product in human AD brains, is synthesized in activated microglial cells and secreted into the extracellular space. The rate of AGE-albumin synthesis in human microglial cells is markedly increased by amyloid-β exposure and oxidative stress. Exogenous AGE-albumin upregulates the receptor protein for AGE (RAGE) and augments calcium influx, leading to apoptosis of human primary neurons. In animal experiments, soluble RAGE (sRAGE), pyridoxamine or ALT-711 prevented Aβ-induced neuronal death in rat brains. Collectively, these results provide evidence for a new mechanism by which microglial cells promote death of neuronal cells through synthesis and secretion of AGE-albumin, thereby likely contributing to neurodegenerative diseases such as AD

Genome editing reveals a role for OCT4 in human embryogenesis.

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.DW was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. NK was supported by the University of Oxford Clarendon Fund. AB was supported by a British Heart Foundation PhD Studentship (FS/11/77/39327). LV was supported by core grant funding from the Wellcome Trust and Medical Research Council (PSAG028). J-SK was supported by the Institute for Basic Science (IBS-R021-D1). Work in the KKN and JMAT labs was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust (FC001120 and FC001193)

Defining Conservation Requirements for the Suweon Treefrog (<i>Dryophytes suweonensis</i>) Using Species Distribution Models

Numerous amphibian species are declining because of habitat loss and fragmentation due to urbanization of landscapes and the construction of roads. This is a mounting threat to species restricted to habitats close to urban areas, such as agricultural wetlands in North East Asia. The Suweon treefrog (Dryophytes suweonensis) falls into the list of species threatened with habitat loss and most populations are under threat of extirpation. Over the last decades, sub-populations have become increasingly disconnected and specifically the density of paved roads has increased around the only site connecting northern and southern Seoul populations. We surveyed this locality in Hojobeol, Siheung, Republic of Korea in 2012, 2015 and 2019 to first confirm the decline in the number of sites where D. suweonensis was present. The second objective was to analyze the habitat characteristics and determine the remaining suitable habitat for D. suweonensis through a species distribution model following the maximum entropy method. Our results show that rice paddy cover and distance from the paved road are the most important factor defining suitable habitat for D. suweonensis. At this locality, uninterrupted rice paddies are a suitable habitat for the species when reaching at least 0.19 km2, with an average distance of 138 ± 93 m2 from the roads. We link the decrease in the number of sites where D. suweonensis is present with the decrease in rice paddy cover, generally replaced by localized infrastructures, greenhouses and habitat fragmentation. Rice paddies should remain connected over a large area for the protection of the remaining populations. In addition, habitat requirements should be integrated in the requisites to designate protected areas