Mammalian mitochondrial DNA (mtDNA) is inherited principally down the maternal line, but the mechanisms involved are not fully understood. Females harboring a mixture of mutant and wild-type mtDNA (heteroplasmy) transmit a varying proportion of mutant mtDNA to their offspring. In humans with mtDNA disorders, the proportion of mutated mtDNA inherited from the mother correlates with disease severity. Rapid changes in allele frequency can occur in a single generation. This could be due to a marked reduction in the number of mtDNA molecules being transmitted from mother to offspring (the mitochondrial genetic bottleneck), to the partitioning of mtDNA into homoplasmic segregating units, or to the selection of a group of mtDNA molecules to re-populate the next generation. Here we show that the partitioning of mtDNA molecules into different cells before and after implantation, followed by the segregation of replicating mtDNA between proliferating primordial germ cells, is responsible for the different levels of heteroplasmy seen in the offspring of heteroplasmic female mice.

The runt related transcription factor CBFA1 (AML3/PEBP2alphaA/RUNX2) regulates expression of several bone- and cartilage-related genes and is required for bone formation in vivo. The gene regulatory mechanisms that control activation and repression of CBFA1 gene transcription during osteoblast differentiation and skeletal development are essential for proper execution of the osteogenic program. We have therefore defined functional contributions of 5' regulatory sequences conserved in rat, mouse and human CBFA1 genes to transcription. Deletion analysis reveals that 0.6 kB of the bone-related rat or mouse CBFA1 promoter (P1, MASNS protein isoform) is sufficient to confer transcriptional activation, and that there are multiple promoter domains which positively and negatively regulate transcription. Progressive deletion of promoter segments between nt -351 and -92 causes a striking 30- to 100-fold combined decrease in promoter activity. Additionally, 5' UTR sequences repress reporter gene transcription 2- to 3-fold. Our data demonstrate that CBFA1 is a principal DNA binding protein interacting with the 5' region of the CBFA1 gene in osseous cells, that there are at least three CBFA1 recognition motifs in the rat CBFA1 promoter, and that there are three tandemly repeated CBFA1 sites within the 5' UTR. We find that forced expression of CBFA1 protein downregulates CBFA1 promoter activity and that a single CBFA1 site is sufficient for transcriptional autosuppression. Thus, our data indicate that the CBFA1 gene is autoregulated in part by negative feedback on its own promoter to stringently control CBFA1 gene expression and function during bone formation.

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...