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Transposon (IS200/IS605)-encoded TnpB proteins are predecessors of class 2 type V CRISPR effectors and have emerged as one of the most compact genome editors identified thus far. Here, we optimized the design of Deinococcus radiodurans (ISDra2) TnpB for application in mammalian cells (TnpBmax), leading to an average 4.4-fold improvement in editing. In addition, we developed variants mutated at position K76 that recognize alternative target-adjacent motifs (TAMs), expanding the targeting range of ISDra2 TnpB. We further generated an extensive dataset on TnpBmax editing efficiencies at 10,211 target sites. This enabled us to delineate rules for on-target and off-target editing and to devise a deep learning model, termed TnpB editing efficiency predictor (TEEP; https://www.tnpb.app ), capable of predicting ISDra2 TnpB guiding RNA (ωRNA) activity with high performance (r > 0.8). Employing TEEP, we achieved editing efficiencies up to 75.3% in the murine liver and 65.9% in the murine brain after adeno-associated virus (AAV) vector delivery of TnpBmax. Overall, the set of tools presented in this study facilitates the application of TnpB as an ultracompact programmable endonuclease in research and therapeutics., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

For gene therapy of the liver, in vivo applications based on adeno-associated virus are the most advanced vectors despite limitations, including low efficacy and episomal loss, potential integration and safety issues, and high production costs. Alternative vectors and/or delivery routes are of high interest. The regenerative ability of the liver bears the potential for ex vivo therapy using liver cell transplantation for disease correction if provided with a selective advantage to expand and replace the existing cell mass. Here we present such treatment of a mouse model of human phenylketonuria (PKU). Primary hepatocytes from wild-type mice were gene modified in vitro (with a lentiviral vector) that carries a gene editing system (CRISPR) to inhibit Cypor. Cypor inactivation confers paracetamol (or acetaminophen) resistance to hepatocytes and thus a growth advantage to eliminate the pre-existing liver cells upon grafting (via the spleen) and exposure to repeated treatment with paracetamol. Grafting Cypor-inactivated wild-type hepatocytes into inbred young adult enu2 (PKU) mice, followed by selective expansion by paracetamol dosing, resulted in replacing up to 5% of cell mass, normalization of blood phenylalanine, and permanent correction of PKU. Hepatocyte transplantation offers thus an armamentarium of novel therapy options for genetic liver defects., (© 2024 The Author(s). Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The success of prime editing depends on the prime editing guide RNA (pegRNA) design and target locus. Here, we developed machine learning models that reliably predict prime editing efficiency. PRIDICT2.0 assesses the performance of pegRNAs for all edit types up to 15 bp in length in mismatch repair-deficient and mismatch repair-proficient cell lines and in vivo in primary cells. With ePRIDICT, we further developed a model that quantifies how local chromatin environments impact prime editing rates., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 ( NCF1 ) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox , an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47 phox and p67 phox , ensuring spatiotemporal and near-physiological transgene expression in myeloid cells., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Vanishing white matter (VWM) is a fatal leukodystrophy caused by recessive mutations in subunits of the eukaryotic translation initiation factor 2B. Currently, there are no effective therapies for VWM. Here, we assessed the potential of adenine base editing to correct human pathogenic VWM variants in mouse models. Using adeno-associated viral vectors, we delivered intein-split adenine base editors into the cerebral ventricles of newborn VWM mice, resulting in 45.9% ± 5.9% correction of the Eif2b5 R191H variant in the cortex. Treatment slightly increased mature astrocyte populations and partially recovered the integrated stress response (ISR) in female VWM animals. This led to notable improvements in bodyweight and grip strength in females; however, locomotor disabilities were not rescued. Further molecular analyses suggest that more precise editing (i.e., lower rates of bystander editing) as well as more efficient delivery of the base editors to deep brain regions and oligodendrocytes would have been required for a broader phenotypic rescue. Our study emphasizes the potential, but also identifies limitations, of current in vivo base-editing approaches for the treatment of VWM or other leukodystrophies., Competing Interests: Declaration of interests G.S. is a scientific advisor to Prime Medicine., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations - the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT - results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells., (© 2024. The Author(s).)

CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N 4 AH and N 5 HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N 3 VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Phenylketonuria (PKU) or hyperphenylalaninemia is considered a paradigm for an inherited (metabolic) liver defect and is, based on murine models that replicate all human pathology, an exemplar model for experimental studies on liver gene therapy. Variants in the PAH gene that lead to hyperphenylalaninemia are never fatal (although devastating if untreated), newborn screening has been available for two generations, and dietary treatment has been considered for a long time as therapeutic and satisfactory. However, significant shortcomings of contemporary dietary treatment of PKU remain. A long list of various gene therapeutic experimental approaches using the classical model for human PKU, the homozygous enu2/2 mouse, witnesses the value of this model to develop treatment for a genetic liver defect. The list of experiments for proof of principle includes recombinant viral (AdV, AAV, and LV) and non-viral (naked DNA or LNP-mRNA) vector delivery methods, combined with gene addition, genome, gene or base editing, and gene insertion or replacement. In addition, a list of current and planned clinical trials for PKU gene therapy is included. This review summarizes, compares, and evaluates the various approaches for the sake of scientific understanding and efficacy testing that may eventually pave the way for safe and efficient human application., (© 2023 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The splicing factor SF3B1 is recurrently mutated in various tumors, including pancreatic ductal adenocarcinoma (PDAC). The impact of the hotspot mutation SF3B1 K700E on the PDAC pathogenesis, however, remains elusive. Here, we demonstrate that Sf3b1 K700E alone is insufficient to induce malignant transformation of the murine pancreas, but that it increases aggressiveness of PDAC if it co-occurs with mutated KRAS and p53. We further show that Sf3b1 K700E already plays a role during early stages of pancreatic tumor progression and reduces the expression of TGF-β1-responsive epithelial-mesenchymal transition (EMT) genes. Moreover, we found that SF3B1 K700E confers resistance to TGF-β1-induced cell death in pancreatic organoids and cell lines, partly mediated through aberrant splicing of Map3k7 . Overall, our findings demonstrate that SF3B1 K700E acts as an oncogenic driver in PDAC, and suggest that it promotes the progression of early stage tumors by impeding the cellular response to tumor suppressive effects of TGF-β., Competing Interests: PS, EI, TM, KM, SA, KV, AK, TT, CS, NA, GR, MS, GS No competing interests declared, (© 2023, Simmler et al.)

Prime editing is a versatile genome editing tool but requires experimental optimization of the prime editing guide RNA (pegRNA) to achieve high editing efficiency. Here we conducted a high-throughput screen to analyze prime editing outcomes of 92,423 pegRNAs on a highly diverse set of 13,349 human pathogenic mutations that include base substitutions, insertions and deletions. Based on this dataset, we identified sequence context features that influence prime editing and trained PRIDICT (prime editing guide prediction), an attention-based bidirectional recurrent neural network. PRIDICT reliably predicts editing rates for all small-sized genetic changes with a Spearman's R of 0.85 and 0.78 for intended and unintended edits, respectively. We validated PRIDICT on endogenous editing sites as well as an external dataset and showed that pegRNAs with high (>70) versus low (<70) PRIDICT scores showed substantially increased prime editing efficiencies in different cell types in vitro (12-fold) and in hepatocytes in vivo (tenfold), highlighting the value of PRIDICT for basic and for translational research applications., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Although dietary fructose is associated with an elevated risk for pancreatic cancer, the underlying mechanisms remain elusive. Here, we report that ketohexokinase (KHK), the rate-limiting enzyme of fructose metabolism, is a driver of PDAC development. We demonstrate that fructose triggers KHK and induces fructolytic gene expression in mouse and human PDAC. Genetic inactivation of Khk C enhances the survival of KPC -driven PDAC even in the absence of high fructose diet. Furthermore, it decreases the viability, migratory capability, and growth of KPC cells in a cell autonomous manner. Mechanistically, we demonstrate that genetic ablation of KHKC strongly impairs the activation of KRAS-MAPK pathway and of rpS6, a downstream target of mTORC signaling. Moreover, overexpression of KHKC in KPC cells enhances the downstream KRAS pathway and cell viability. Our data provide new insights into the role of KHK in PDAC progression and imply that inhibiting KHK could have profound implications for pancreatic cancer therapy., Competing Interests: M.S. is a member of the Scientific Advisory Board of Alnylam Pharmaceuticals. All other authors declare no conflict of interest relating to this study., (© 2023 The Authors.)

Mutations in the splicing factor SF3B1 are frequently occurring in various cancers and drive tumor progression through the activation of cryptic splice sites in multiple genes. Recent studies also demonstrate a positive correlation between the expression levels of wild-type SF3B1 and tumor malignancy. Here, we demonstrate that SF3B1 is a hypoxia-inducible factor (HIF)-1 target gene that positively regulates HIF1 pathway activity. By physically interacting with HIF1α, SF3B1 facilitates binding of the HIF1 complex to hypoxia response elements (HREs) to activate target gene expression. To further validate the relevance of this mechanism for tumor progression, we show that a reduction in SF3B1 levels via monoallelic deletion of Sf3b1 impedes tumor formation and progression via impaired HIF signaling in a mouse model for pancreatic cancer. Our work uncovers an essential role of SF3B1 in HIF1 signaling, thereby providing a potential explanation for the link between high SF3B1 expression and aggressiveness of solid tumors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDA) is an inherently immune cell deprived tumor, characterized by desmoplastic stroma and suppressive immune cells. Here we systematically dissect PDA intrinsic mechanisms of immune evasion by in vitro and in vivo CRISPR screening, and identify Vps4b and Rnf31 as essential factors required for escaping CD8 + T cell killing. For Vps4b we find that inactivation impairs autophagy, resulting in increased accumulation of CD8 + T cell-derived granzyme B and subsequent tumor cell lysis. For Rnf31 we demonstrate that it protects tumor cells from TNF-mediated caspase 8 cleavage and subsequent apoptosis induction, a mechanism that is conserved in human PDA organoids. Orthotopic transplantation of Vps4b- or Rnf31 deficient pancreatic tumors into immune competent mice, moreover, reveals increased CD8 + T cell infiltration and effector function, and markedly reduced tumor growth. Our work uncovers vulnerabilities in PDA that might be exploited to render these tumors more susceptible to the immune system., (© 2022. The Author(s).)

Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced Sp Cas9 prime editor (PE) lacking the RNaseH domain (PE2 Δ RnH ) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2 Δ RnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pah enu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 10 14 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.

Vanishing white matter (VWM) is a leukodystrophy caused by recessive variants in subunits of eIF2B. At present, no curative treatment is available and patients often die at young age. Due to its monogenic nature, VWM is a promising candidate for the development of CRISPR/Cas9-mediated gene therapy. Here we tested a dual-AAV approach in VWM mice encoding CRISPR/Cas9 and a DNA donor template to correct a pathogenic variant in Eif2b5 . We performed sequencing analysis to assess gene correction rates and examined effects on the VWM phenotype, including motor behavior. Sequence analysis demonstrated that over 90% of CRISPR/Cas9-induced edits at the targeted locus are insertion or deletion (indel) mutations, rather than precise corrections from the DNA donor template by homology-directed repair. Around half of the CRISPR/Cas9-treated animals died prematurely. VWM mice showed no improvement in motor skills, weight, or neurological scores at 7 months of age, and CRISPR/Cas9-treated controls displayed an induced VWM phenotype. In conclusion, CRISPR/Cas9-induced DNA double-strand breaks (DSBs) at the Eif2b5 locus did not lead to sufficient correction of the VWM variant. Moreover, indel formation in Eif2b5 induced an exacerbated VWM phenotype. Therefore, DSB-independent strategies like base- or prime editing might better suited for VWM correction., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2 . We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo . The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks., Competing Interests: Declaration of interests Janette Beshay, Hagen Klett and Julia Schueler are employees of Charles River Research Services Germany GmbH. The other authors declare no competing interests.

The CRISPR/Cas technology has revolutionized forward genetic screening, and thereby facilitated genetic dissection of cellular processes and pathways. TGF-β signaling is a highly conserved cascade involved in development, regeneration, and diseases such as cancer. Even though many core components of the signaling cascade have already been described, several context-dependent pathway modulators remain unknown. To address this knowledge gap, we have recently developed a CRISPR screening approach for identifying TGF-β pathway regulators in three-dimensional organoid culture systems. Here, we provide a detailed protocol describing this approach in human intestinal organoids. With adaptations, this screening method could also be applied to other organoid types, and to other signaling cascades such as EGF or WNT signaling, thereby uncovering important mechanism in regeneration and disease., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor that is almost uniformly lethal in humans. Activating mutations of KRAS are found in >90% of human PDACs and are sufficient to promote acinar-to-ductal metaplasia (ADM) during tumor initiation. The roles of miRNAs in oncogenic Kras-induced ADM are incompletely understood., Methods: The Ptf1a Cre/+ LSL-Kras G12D/+ and Ptf1a Cre/+ LSL-Kras G12D/+ LSL-p53R172H/ + and caerulein-induced acute pancreatitis mice models were used. mir-802 was conditionally ablated in acinar cells to study the function of miR-802 in ADM., Results: We show that miR-802 is a highly abundant and acinar-enriched pancreatic miRNA that is silenced during early stages of injury or oncogenic Kras G12D -induced transformation. Genetic ablation of mir-802 cooperates with Kras G12D by promoting ADM formation. miR-802 deficiency results in de-repression of the miR-802 targets Arhgef12, RhoA, and Sdc4, activation of RhoA, and induction of the downstream RhoA effectors ROCK1, LIMK1, COFILIN1, and EZRIN, thereby increasing F-actin rearrangement. mir-802 ablation also activates SOX9, resulting in augmented levels of ductal and attenuated expression of acinar identity genes. Consistently with these findings, we show that this miR-802-RhoA-F-actin network is activated in biopsies of pancreatic cancer patients and correlates with poor survival., Conclusions: We show miR-802 suppresses pancreatic cancer initiation by repressing oncogenic Kras-induced ADM. The role of miR-802 in ADM fills the gap in our understanding of oncogenic Kras-induced F-actin reorganization, acinar reprogramming, and PDAC initiation. Modulation of the miR-802-RhoA-F-actin network may be a new strategy to interfere with pancreatic carcinogenesis., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA., Competing Interests: L.V. and G.S. have filed a patent application based on these constructs., (© 2021 The Author(s).)

Base editors are chimeric ribonucleoprotein complexes consisting of a DNA-targeting CRISPR-Cas module and a single-stranded DNA deaminase. They enable transition of C•G into T•A base pairs and vice versa on genomic DNA. While base editors have great potential as genome editing tools for basic research and gene therapy, their application has been hampered by a broad variation in editing efficiencies on different genomic loci. Here we perform an extensive analysis of adenine- and cytosine base editors on a library of 28,294 lentivirally integrated genetic sequences and establish BE-DICT, an attention-based deep learning algorithm capable of predicting base editing outcomes with high accuracy. BE-DICT is a versatile tool that in principle can be trained on any novel base editor variant, facilitating the application of base editing for research and therapy., (© 2021. The Author(s).)

Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle-based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases., (© 2021. The Author(s).)

The intestinal epithelium is a complex structure that integrates digestive, immunological, neuroendocrine, and regenerative functions. Epithelial homeostasis is maintained by a coordinated cross-talk of different epithelial cell types. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here we show that the intestine-enriched miR-802 is a central regulator of intestinal epithelial cell proliferation, Paneth cell function, and enterocyte differentiation. Genetic ablation of mir-802 in the small intestine of mice leads to decreased glucose uptake, impaired enterocyte differentiation, increased Paneth cell function and intestinal epithelial proliferation. These effects are mediated in part through derepression of the miR-802 target Tmed9, a modulator of Wnt and lysozyme/defensin secretion in Paneth cells, and the downstream Wnt signaling components Fzd5 and Tcf4. Mutant Tmed9 mice harboring mutations in miR-802 binding sites partially recapitulate the augmented Paneth cell function of mice lacking miR-802. Our study demonstrates a broad miR-802 network that is important for the integration of signaling pathways of different cell types controlling epithelial homeostasis in the small intestine.

Intra-tumor hypoxia is a common feature in many solid cancers. Although transcriptional targets of hypoxia-inducible factors (HIFs) have been well characterized, alternative splicing or processing of pre-mRNA transcripts which occurs during hypoxia and subsequent HIF stabilization is much less understood. Here, we identify many HIF-dependent alternative splicing events after whole transcriptome sequencing in pancreatic cancer cells exposed to hypoxia with and without downregulation of the aryl hydrocarbon receptor nuclear translocator (ARNT), a protein required for HIFs to form a transcriptionally active dimer. We correlate the discovered hypoxia-driven events with available sequencing data from pan-cancer TCGA patient cohorts to select a narrow set of putative biologically relevant splice events for experimental validation. We validate a small set of candidate HIF-dependent alternative splicing events in multiple human gastrointestinal cancer cell lines as well as patient-derived human pancreatic cancer organoids. Lastly, we report the discovery of a HIF-dependent mechanism to produce a hypoxia-dependent, long and coding isoform of the UDP-N-acetylglucosamine transporter SLC35A3., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Base editors are RNA-programmable deaminases that enable precise single-base conversions in genomic DNA. However, off-target activity is a concern in the potential use of base editors to treat genetic diseases. Here, we report unbiased analyses of transcriptome-wide and genome-wide off-target modifications effected by cytidine base editors in the liver of mice with phenylketonuria. The intravenous delivery of intein-split cytidine base editors by dual adeno-associated viruses led to the repair of the disease-causing mutation without generating off-target mutations in the RNA and DNA of the hepatocytes. Moreover, the transient expression of a cytidine base editor mRNA and a relevant single-guide RNA intravenously delivered by lipid nanoparticles led to ~21% on-target editing and to the reversal of the disease phenotype; there were also no detectable transcriptome-wide and genome-wide off-target edits. Our findings support the feasibility of therapeutic cytidine base editing to treat genetic liver diseases.

Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at the industrial scale. Here, we report the fabrication of microengineered cell culture devices and scalable and automated methods for suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate. The absence of a solid matrix substantially reduces organoid heterogeneity, which we show for mouse and human gastrointestinal organoids. We use the devices to screen for anticancer drug candidates with patient-derived colorectal cancer organoids, and apply high-content image-based phenotypic analyses to reveal insights into mechanisms of drug action. The scalable organoid-culture technology should facilitate the use of organoids in drug development and diagnostics.

Intestinal epithelial organoids established from gut tissue have become a widely used research tool. However, it remains unclear how environmental cues, divergent microbiota composition and other sources of variation before, during and after establishment confound organoid properties, and how these properties relate to the original tissue. While environmental influences cannot be easily addressed in human organoids, mice offer a controlled assay-system. Here, we probed the effect of donor microbiota differences, previously identified as a confounding factor in murine in vivo studies, on organoids. We analysed the proteomes and transcriptomes of primary organoid cultures established from two colonised and one germ-free mouse colony of C57BL/6J genetic background, and compared them to their tissue of origin and commonly used cell lines. While an imprint of microbiota-exposure was observed on the proteome of epithelial samples, the long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead, stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts and closely mimicked expression patterns in the gut epithelium. This included the inflammasome components ASC, Naip1-6, Nlrc4 and Caspase-1, which were highly expressed in all organoids compared to the reference cell line m-IC c12 or mouse embryonic fibroblasts. Taken together, these results reveal that the donor microbiota has little effect on the organoid phenotype and suggest that organoids represent a more suitable culture model than immortalised cell lines, in particular for studies of intestinal epithelial inflammasomes., (© 2020 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-β-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-β resistance and attenuate TGF-β target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models., Competing Interests: Declaration of Interests H.C. is an inventor on applications/patents related to organoid technology. The other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Phenylketonuria (PKU) is considered to be a paradigm for a monogenic metabolic disorder but was never thought to be a primary application for human gene therapy due to established alternative treatment. However, somewhat unanticipated improvement in neuropsychiatric outcome upon long-term treatment of adults with PKU with enzyme substitution therapy might slowly change this assumption. In parallel, PKU was for a long time considered to be an excellent test system for experimental gene therapy of a Mendelian autosomal recessive defect of the liver due to an outstanding mouse model and the easy to analyze and well-defined therapeutic end point, that is, blood l-phenylalanine concentration. Lifelong treatment by targeting the mouse liver (or skeletal muscle) was achieved using different approaches, including (1) recombinant adeno-associated viral (rAAV) or nonviral naked DNA vector-based gene addition, (2) genome editing using base editors delivered by rAAV vectors, and (3) by delivering rAAVs for promoter-less insertion of the PAH -cDNA into the Pah locus. In this article we summarize the gene therapeutic attempts of correcting a mouse model for PKU and discuss the future implications for human gene therapy.

The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating small insertion/deletion (indel) mutations, or by homology-directed repair (HDR). If ectopic donor templates are provided, the latter mechanism allows editing with single-nucleotide precision. The preference of mammalian cells to repair DSBs by NHEJ rather than HDR, however, limits the potential of CRISPR-Cas9 for applications where precise editing is needed. To enhance the efficiency of DSB repair by HDR from donor templates, we recently engineered a CRISPR-Cas9 system where the template DNA is bound to the Cas9 enzyme. In short, single-stranded oligonucleotides were labeled with O6-benzylguanine (BG), and covalently linked to a Cas9-SNAP-tag fusion protein to form a ribonucleoprotein-DNA (RNPD) complex consisting of the Cas9 nuclease, the sgRNA, and the repair template. Here, we provide a detailed protocol how to generate O6-benzylguanine (BG)-linked DNA repair templates, produce recombinant Cas9-SNAP-tag fusion proteins, in vitro transcribe single guide RNAs, and transfect RNPDs into various mammalian cells., Competing Interests: Competing interests The authors declare no competing interests.

CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah) enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU) 1 , using recently developed CRISPR-Cas-associated base editors 2-4 . These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pah enu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pah enu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.

Epithelial organoids are simplified models of organs grown in vitro from embryonic and adult stem cells. They are widely used to study organ development and disease, and enable drug screening in patient-derived primary tissues. Current protocols, however, rely on animal- and tumor-derived basement membrane extract (BME) as a 3D scaffold, which limits possible applications in regenerative medicine. This prompted us to study how organoids interact with their matrix, and to develop a well-defined hydrogel that supports organoid generation and growth. It is found that soft fibrin matrices provide suitable physical support, and that naturally occurring Arg-Gly-Asp (RGD) adhesion domains on the scaffold, as well as supplementation with laminin-111, are key parameters required for robust organoid formation and expansion. The possibility to functionalize fibrin via factor XIII-mediated anchoring also allows to covalently link fluorescent nanoparticles to the matrix for 3D traction force microscopy. These measurements suggest that the morphogenesis of budding intestinal organoids results from internal pressure combined with higher cell contractility in the regions containing differentiated cells compared to the regions containing stem cells. Since the fibrin/laminin matrix supports long-term expansion of all tested murine and human epithelial organoids, this hydrogel can be widely used as a defined equivalent to BME., (© 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second most frequent cause of cancer-related mortality worldwide. The multikinase inhibitor sorafenib is the only treatment option for advanced HCC. Due to tumor heterogeneity, its efficacy greatly varies between patients and is limited due to adverse effects and drug resistance. Current in vitro models fail to recapitulate key features of HCCs. We report the generation of long-term organoid cultures from tumor needle biopsies of HCC patients with various etiologies and tumor stages. HCC organoids retain the morphology as well as the expression pattern of HCC tumor markers and preserve the genetic heterogeneity of the originating tumors. In a proof-of-principle study, we show that liver cancer organoids can be used to test sensitivity to sorafenib. In conclusion, organoid models can be derived from needle biopsies of liver cancers and provide a tool for developing tailored therapies., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired., Competing Interests: NS, FR, HL, CB, KB, YL, DN, MR, CC, JH, MJ, GS No competing interests declared, (© 2018, Savic et al.)

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.

Targeted nucleases are widely used as tools for genome editing. Two years ago the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease was used for the first time, and since then has largely revolutionized the field. The tremendous success of the CRISPR/Cas9 genome editing tool is powered by the ease design principle of the guide RNA that targets Cas9 to the desired DNA locus, and by the high specificity and efficiency of CRISPR/Cas9-generated DNA breaks. Several studies recently used CRISPR/Cas9 to successfully modulate disease-causing alleles in vivo in animal models and ex vivo in somatic and induced pluripotent stem cells, raising hope for therapeutic genome editing in the clinics. In this review, we will summarize and discuss such preclinical CRISPR/Cas9 gene therapy reports., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The CRISPR/Cas9 system is an RNA-guided genome-editing tool that has been recently developed based on the bacterial CRISPR-Cas immune defense system. Due to its versatility and simplicity, it rapidly became the method of choice for genome editing in various biological systems, including mammalian cells. Here we describe a protocol for CRISPR/Cas9-mediated genome editing in murine small intestinal organoids, a culture system in which somatic stem cells are maintained by self-renewal, while giving rise to all major cell types of the intestinal epithelium. This protocol allows the study of gene function in intestinal epithelial homeostasis and pathophysiology and can be extended to epithelial organoids derived from other internal mouse and human organs.

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.

Paneth cells (PCs) are terminally differentiated, highly specialized secretory cells located at the base of the crypts of Lieberkühn in the small intestine. Besides their antimicrobial function, PCs serve as a component of the intestinal stem cell niche. By secreting granules containing bactericidal proteins like defensins/cryptdins and lysozyme, PCs regulate the microbiome of the gut. Here we study the control of PC degranulation in primary epithelial organoids in culture. We show that PC degranulation does not directly occur upon stimulation with microbial antigens or bacteria. In contrast, the pro-inflammatory cytokine Interferon gamma (IFN-γ) induces rapid and complete loss of granules. Using live cell imaging, we show that degranulation is coupled to luminal extrusion and death of PCs. Transfer of supernatants from in vitro stimulated iNKT cells recapitulates degranulation in an IFN-γ-dependent manner. Furthermore, endogenous IFN-γ secretion induced by anti-CD3 antibody injection causes Paneth loss and release of goblet cell mucus. The identification of IFN-γ as a trigger for degranulation and extrusion of PCs establishes a novel effector mechanism by which immune responses may regulate epithelial status and the gut microbiome., (© 2014 Farin et al.)

Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Under previously developed culture conditions, mouse and human intestinal epithelia can be cultured and expanded over long periods. These so-called organoids recapitulate the three-dimensional architecture of the gut epithelium, and consist of all major intestinal cell types. One key advantage of these ex vivo cultures is their accessibility to live imaging. So far the establishment of transgenic fluorescent reporter organoids has required the generation of transgenic mice, a laborious and time-consuming process, which cannot be extended to human cultures. Here we present a transfection protocol that enables the generation of recombinant mouse and human reporter organoids using BAC (bacterial artificial chromosome) technology.