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...

Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding β‐catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.

CRISPR/Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences in human organoids awaits robust knock-in approaches. Here, we describe CRISPR/Cas9-mediated Homologyindependent Organoid Transgenesis (CRISPR-HOT), which allows efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences at desired loci, without the necessity to inactivate TP53 in untransformed cells, previously used to increase HDR-mediated knock-in. CRISPR-HOT was employed to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter labelling the mitotic spindle by tagged tubulin and the cell membrane by tagged E-cadherin uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed TP53 involvement in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.

15Prime editing is a novel genome editing technology using fusion proteins of Cas9-nickase 16 and reverse transcriptase, that holds promise to correct a wide variety of genetic defects. 17We succeeded in efficient prime editing and functional recovery of disease-causing 18 mutations in patient-derived liver and intestinal stem cell organoids. 19 20 Main 21The development of gene-editing therapies to treat monogenic diseases has long been an essential 22 goal of CRISPR/Cas9 research. Cas9-mediated homology-directed repair (HDR) can create all desired 23 base substitutions, insertions and deletions (indels). However, HDR relies on introduction of double-24 stranded DNA breaks, is inefficient and error-prone 1,2 . Base editing, that uses Cas9-nickases fused to 25 DNA-modifying enzymes, is more efficient and accurate than HDR, but can only correct four out of 26 twelve point mutations and no small insertions and deletions. Furthermore, base editing requires a 27 suitable protospacer adjacent motif (PAM) and the absence of co-editable nucleotides 3 . 28Prime editing combines a nicking-Cas9-reverse transcriptase fusion protein (PE2) with a prime 29 editing guide RNA (pegRNA) containing the desired edit. The pegRNA-spacer guides the formation of 30 a nick in the targeted DNA strand. The pegRNA-extension binds to this nicked strand and instructs 31 the synthesis of an edited DNA flap. This edited flap is then integrated by DNA repair mechanisms, 32 which can be enhanced by simultaneous nicking of the non-edited strand (Supplementary Fig. 1) 4 . 33Prime editing has been applied in human cancer cell lines and plant cells, but not in human disease 34 models 4,5,6 . Adult stem cell-derived organoids exhibit important functional properties of organs, 35allowing modeling of monogenic diseases 7 . We set out to develop and test prime editing in primary 36 patient-derived organoids. 38We first optimized prime editing for organoid cells using deletions and single-nucleotide 39 substitutions previously performed in HEK293T cells 4 . The non-edited strand was nicked by a second 40 'nicking sgRNA' to enhance editing (PE3). Our optimized protocol consisted of co-transfection of 41 prime edit plasmids with a GFP-reporter plasmid allowing selection and subsequent clonal expansion 42 of transfected cells. Prime editing of intestinal and ductal liver organoids resulted in efficient (>50%) 43 deletion of 5 nucleotides in HEK3 ( Fig. 1b and Supplementary Fig. 2). Furthermore, we were able to 44 induce a transversion mutation located 26 nucleotides downstream of the nick with substantial 45 efficiency (20%) (Fig. 1c). These results show successful prime editing of primary stem cells with 46 similar efficiency as observed in human cancer cell lines.Next, we targeted the Wnt-pathway intermediate ß-catenin (CTNNB1) in organoids. Activating 48 carcinogenic CTNNB1 mutations are found in ±40% of hepatocellular carcinoma, resulting in Wnt-49 signaling independent of exogenous stimuli 8 . We designed PE3 plasmids, containing pegRNA-50 extensions wi...