rganoids can be generated by guided differentiation of induced pluripotent stem cells and embryonic stem cells, or from cells isolated from adult tissues 1 . Adult stem cell (ASC)-derived organoids are self-organizing structures that recapitulate aspects of cellular composition, three-dimensional (3D) architecture and functionality of the different epithelial tissues from which they originate, while maintaining genomic stability 2,3 . The possibility to derive organoids from genetically modified mouse lines, especially knock-in models, has enabled the generation of engineered mouse organoids that have been used as versatile in vitro tools to answer various biological questions [4][5][6][7][8][9][10] .The generation of engineered human ASC-derived organoids requires that efficient strategies for in vitro genome editing are applied after the lines have been established. CRISPR-Cas9 technology has simplified genetic engineering considerably. To date, these approaches were largely limited to the non-homologous end joining (NHEJ)-mediated introduction of indels into the endogenous loci of organoids, leading to gene mutations [11][12][13][14] . By harnessing the HDR pathway, a single-base substitution was introduced to correct the CFTR locus in cystic fibrosis intestinal organoids 15 , and a few human ASC-organoid knock-in reporter lines have been generated, but mostly in colon cancer organoids [16][17][18] .Knock-in using HDR takes advantage of a mechanism used by cells to repair double-stranded breaks (DSBs). Such breaks can be introduced at specific sites using CRISPR-Cas9. HDR is the most commonly used approach for targeted insertion, but this process is inefficient and requires cells to be in S phase 19,20 . Furthermore, HDR requires that the donor plasmid is cloned, owing to the necessity for the presence of homology arms specific to each gene (Fig. 1a). Recent studies have shown that CRISPR-induced DSBs activate the TP53 damage response and induce a transient cell-cycle arrest in untransformed cells 21 . Permanent or transient inactivation of TP53 increases HDR-mediated knock-in in pluripotent and hematopoietic stem cells 22,23 . Thus, given the demand for novel methods to improve HDR efficiency, inhibition of TP53 was suggested as a potential solution to overcome the low efficiency of HDR-mediated knock-in in untransformed cells 23 .NHEJ, another key DNA repair system, is active in all cell cycle phases 20 and, by ligating DNA ends, does not require regions of homology (Fig. 1a). As NHEJ is generally believed to be error prone, it is not widely used for precision transgene insertion. However, it has been suggested that NHEJ can be fundamentally accurate and can religate DNA ends without errors 24,25 . Indeed, a handful of studies have exploited NHEJ to ensure the targeted insertion of exogenous DNA into zebrafish 26 , mouse 27 , immortalized human cell lines 28,29 and embryonic stem cells 30 . Here we leverage NHEJ-mediated knock-in for use in the human organoid field-an approach named CRISPR-HOT-as a versatile...

22While the spatiotemporal structure of the genome is crucial to its biological function, many basic questions 23 remain unanswered on the morphology and segregation of chromosomes. Here, we experimentally show in 24 Escherichia coli that spatial confinement plays a dominant role in determining both the chromosome size 25 and position. In non-dividing cells with lengths up to 10 times normal, single chromosomes are observed 26 to expand more than 4 fold in size, an effect only modestly influenced by deletions of various nucleoid-27 associated proteins. Chromosomes show pronounced internal dynamics but exhibit a robust positioning 28 where single nucleoids reside strictly at mid-cell, while two nucleoids self-organize at ¼ and ¾ cell 29 positions. Molecular dynamics simulations of model chromosomes recapitulate these phenomena and 30 indicate that these observations can be attributed to depletion effects induced by cytosolic crowders. These 31 findings highlight boundary confinement as a key causal factor that needs to be considered for 32 understanding chromosome organization. 33 34 Key words 35 36

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on nonspecific DNA [1][2][3][4][5] . Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 µs. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.Sequence-specific binding and recognition of DNA target sites by proteins such as transcription factors, polymerases, and DNA-modifying enzymes is at the core of cellular information processing. However, the 'target search problem' of how to rapidly yet accurately find a specific target sequence remains incompletely understood. One aspect of preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Grafted mesenchymal stem cells (MSCs) yield neuroprotection in preclinical stroke models by secreting extracellular vesicles (EVs). The neuroprotective cargo of EVs, however, has not yet been identified. To investigate such cargo and its underlying mechanism, primary neurons were exposed to oxygen‐glucose‐deprivation (OGD) and cocultured with adipose‐derived MSCs (ADMSCs) or ADMSC‐secreted EVs. Under such conditions, both ADMSCs and ADMSC‐secreted EVs significantly reduced neuronal death. Screening for signalling cascades being involved in the interaction between ADMSCs and neurons revealed a decreased autophagic flux as well as a declined p53‐BNIP3 activity in neurons receiving either treatment paradigm. However, the aforementioned effects were reversed when ADMSCs were pretreated with the inhibitor of exosomal secretion GW4869 or when Hrs was knocked down. In light of miR‐25‐3p being the most highly expressed miRNA in ADMSC‐EVs interacting with the p53 pathway, further in vitro work focused on this pathway. Indeed, a miR‐25‐3p oligonucleotide mimic reduced cell death, whereas the anti‐oligonucleotide increased autophagic flux and cell death by modulating p53‐BNIP3 signalling in primary neurons exposed to OGD. Likewise, native ADMSC‐EVs but not EVs obtained from ADMSCs pretreated with the anti‐miR‐25‐3p oligonucleotide (ADMSC‐EVsanti‐miR‐25‐3p) confirmed the aforementioned in vitro observations in C57BL/6 mice exposed to cerebral ischemia. The infarct size was reduced, and neurological recovery was increased in mice treated with native ADMSC‐EVs when compared to ADMSC‐EVsanti‐miR‐25‐3p. ADMSCs induce neuroprotection by improved autophagic flux through secreted EVs containing miR‐25‐3p. Hence, our work uncovers a novel key factor in naturally secreted ADMSC‐EVs for the regulation of autophagy and induction of neuroprotection in a preclinical stroke model.