Modeling Human Digestive Diseases With CRISPR-Cas9–Modified Organoids

Insights into the stem cell niche have allowed researchers to cultivate adult tissue stem cells as organoids that display structural and phenotypic features of healthy and diseased epithelial tissues. Organoids derived from patients’ tissues are used as models of disease and to test drugs. CRISPR-Cas9 technology can be used to genetically engineer organoids for studies of monogenic diseases and cancer. We review the derivation of organoids from human gastrointestinal tissues and how CRISPR-Cas9 technology can be used to study these organoids. We discuss burgeoning technologies that are broadening our understanding of diseases of the digestive system. Insights into the stem cell niche have allowed researchers to cultivate adult tissue stem cells as organoids that display structural and phenotypic features of healthy and diseased epithelial tissues. Organoids derived from patients’ tissues are used as models of disease and to test drugs. CRISPR-Cas9 technology can be used to genetically engineer organoids for studies of monogenic diseases and cancer. We review the derivation of organoids from human gastrointestinal tissues and how CRISPR-Cas9 technology can be used to study these organoids. We discuss burgeoning technologies that are broadening our understanding of diseases of the digestive system. Heritable or acquired genetic aberrations underlie numerous human diseases affecting gastrointestinal (GI) and other organ systems, either by directly compromising vital functions or by working in concert with environmental or epigenetic effects.1Feinberg A.P. The key role of epigenetics in human disease prevention and mitigation.N Engl J Med. 2018; 378: 1323-1334Crossref PubMed Scopus (94) Google Scholar, 2Rappaport S.M. Smith M.T. Epidemiology. Environment and disease risks.Science. 2010; 330: 460-461Crossref PubMed Scopus (0) Google Scholar Genome-wide approaches, including linkage analysis and genome-wide association studies, have identified genetic lesions and variations related to human disease phenotypes. Recent advances in DNA sequencing technology have further accelerated the mining of pathogenic gene abnormalities, especially in the context of cancer.3Garraway L.A. Lander E.S. Lessons from the cancer genome.Cell. 2013; 153: 17-37Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar Such cross-sectional investigations have substantiated genotype–phenotype correlations underlying human diseases. Nonetheless, phenotypic confirmation by prospective genetic modification is crucial to establish the causality that provides the mechanistic understanding of disease pathophysiology, and to develop novel therapeutic strategies. To manipulate genetic function and examine the consequent phenotype, researchers have conventionally opted for short hairpin RNA–mediated gene knockdown or for overexpression experiments in human cell lines or for genetically engineered rodents. Although these strategies have contributed significantly to the understanding of a plethora of genotype–phenotype correlations, there exist caveats regarding the translatability of the outcomes to the human condition. Gene knockdown experiments are often complicated by low knockdown efficiency and off-target effects, while gene overexpression typically produces transcripts at a non-physiological level. Moreover, most human cell lines derived from embryonic or cancer cells do not fully capture phenotypic and histologic attributes of human tissues. Establishing genetically engineered mouse models is a time- and effort-intensive practice, and the interspecies differences between human and rodents cannot be overlooked. Two novel technologies that—in combination—potentially override these limitations have emerged recently. CRISPR-Cas9 genome editing technology offers introduction of DNA double-strand breaks at specific genomic loci and enables gene knockout and knock-in more efficiently than any other class of genome editors,4Komor A.C. Badran A.H. Liu D.R. CRISPR-based technologies for the manipulation of eukaryotic genomes.Cell. 2017; 168: 20-36Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar and the engineering of Cas9 has broadened its capability to other applications, including base editing, epigenome editing, and gene expression manipulation.5Adli M. The CRISPR tool kit for genome editing and beyond.Nat Commun. 2018; 9: 1911Crossref PubMed Scopus (173) Google Scholar Organoid technology allows long-term 3-dimensional (3D) reconstruction of various tissues from stem cells with the preservation of tissue phenotypes.6Clevers H. Modeling development and disease with organoids.Cell. 2016; 165: 1586-1597Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar Since the first report on mouse intestinal organoids,7Sato T. Vries R.G. Snippert H.J. et al.Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (2436) Google Scholar its application has rapidly extended to other GI and non-GI tissues of mice and man.8Jung P. Sato T. Merlos-Suarez A. et al.Isolation and in vitro expansion of human colonic stem cells.Nat Med. 2011; 17: 1225-1227Crossref PubMed Scopus (359) Google Scholar, 9Sato T. Stange D.E. Ferrante M. et al.Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (1053) Google Scholar, 10Barker N. Huch M. Kujala P. et al.Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.Cell Stem Cell. 2010; 6: 25-36Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar, 11Bartfeld S. Bayram T. van de Wetering M. et al.In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection.Gastroenterology. 2015; 148: 126-136 e6Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 12Huch M. Bonfanti P. Boj S.F. et al.Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.EMBO J. 2013; 32: 2708-2721Crossref PubMed Scopus (279) Google Scholar, 13Boj S.F. Hwang C.I. Baker L.A. et al.Organoid models of human and mouse ductal pancreatic cancer.Cell. 2015; 160: 324-338Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 14Huch M. Dorrell C. Boj S.F. et al.In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.Nature. 2013; 494: 247-250Crossref PubMed Scopus (624) Google Scholar, 15Huch M. Gehart H. van Boxtel R. et al.Long-term culture of genome-stable bipotent stem cells from adult human liver.Cell. 2015; 160: 299-312Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 16Lugli N. Kamileri I. Keogh A. et al.R-spondin 1 and noggin facilitate expansion of resident stem cells from non-damaged gallbladders.EMBO Rep. 2016; 17: 769-779Crossref PubMed Google Scholar, 17Sampaziotis F. Justin A.W. Tysoe O.C. et al.Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.Nat Med. 2017; 23: 954-963Crossref PubMed Scopus (50) Google Scholar, 18DeWard A.D. Cramer J. Lagasse E. Cellular heterogeneity in the mouse esophagus implicates the presence of a nonquiescent epithelial stem cell population.Cell Rep. 2014; 9: 701-711Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 19Jiang M. Li H. Zhang Y. et al.Transitional basal cells at the squamous-columnar junction generate Barrett's oesophagus.Nature. 2017; 550: 529-533Crossref PubMed Scopus (57) Google Scholar, 20Kasagi Y. Chandramouleeswaran P.M. Whelan K.A. et al.The esophageal organoid system reveals functional interplay between notch and cytokines in reactive epithelial changes.Cell Mol Gastroenterol Hepatol. 2018; 5: 333-352Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 21Karthaus W.R. Iaquinta P.J. Drost J. et al.Identification of multipotent luminal progenitor cells in human prostate organoid cultures.Cell. 2014; 159: 163-175Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 22Chua C.W. Shibata M. Lei M. et al.Single luminal epithelial progenitors can generate prostate organoids in culture.Nat Cell Biol. 2014; 16 (951–61, 1–4)Crossref PubMed Scopus (125) Google Scholar, 23Jarde T. Lloyd-Lewis B. Thomas M. et al.Wnt and Neuregulin1/ErbB signalling extends 3D culture of hormone responsive mammary organoids.Nat Commun. 2016; 7: 13207Crossref PubMed Scopus (25) Google Scholar, 24Sachs N. de Ligt J. Kopper O. et al.A living biobank of breast cancer organoids captures disease heterogeneity.Cell. 2018; 172: 373-386 e10Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 25Turco M.Y. Gardner L. Hughes J. et al.Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium.Nat Cell Biol. 2017; 19: 568-577Crossref PubMed Scopus (70) Google Scholar, 26Kessler M. Hoffmann K. Brinkmann V. et al.The Notch and Wnt pathways regulate stemness and differentiation in human fallopian tube organoids.Nat Commun. 2015; 6: 8989Crossref PubMed Google Scholar, 27Ren W. Lewandowski B.C. Watson J. et al.Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo.Proc Natl Acad Sci U S A. 2014; 111: 16401-16406Crossref PubMed Scopus (63) Google Scholar, 28Maimets M. Rocchi C. Bron R. et al.Long-term in vitro expansion of salivary gland stem cells driven by Wnt signals.Stem Cell Reports. 2016; 6: 150-162Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 29Rock J.R. Onaitis M.W. Rawlins E.L. et al.Basal cells as stem cells of the mouse trachea and human airway epithelium.Proc Natl Acad Sci U S A. 2009; 106: 12771-12775Crossref PubMed Scopus (686) Google Scholar Currently, the term organoid broadly refers to organotypic structures generated from somatic stem cells, as well as those derived from pluripotent cells by directed differentiation.30Lancaster M.A. Knoblich J.A. Organogenesis in a dish: modeling development and disease using organoid technologies.Science. 2014; 345: 1247125Crossref PubMed Scopus (705) Google Scholar The usage of pluripotent stem cell–derived organoids has been described comprehensively elsewhere.6Clevers H. Modeling development and disease with organoids.Cell. 2016; 165: 1586-1597Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 31Fatehullah A. Tan S.H. Barker N. Organoids as an in vitro model of human development and disease.Nat Cell Biol. 2016; 18: 246-254Crossref PubMed Scopus (419) Google Scholar, 32Dedhia P.H. Bertaux-Skeirik N. Zavros Y. et al.Organoid models of human gastrointestinal development and disease.Gastroenterology. 2016; 150: 1098-1112Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 33McCauley H.A. Wells J.M. Pluripotent stem cell-derived organoids: using principles of developmental biology to grow human tissues in a dish.Development. 2017; 144: 958-962Crossref PubMed Scopus (50) Google Scholar, 34Eicher A.K. Berns H.M. Wells J.M. Translating developmental principles to generate human gastric organoids.Cell Mol Gastroenterol Hepatol. 2018; 5: 353-363Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar In this review article, we specifically focus on the utility of healthy/diseased tissue stem cell organoids (hereafter simply called organoids for brevity) and discuss how they can be coupled with the CRISPR-Cas9 technology to model and study human GI diseases. The organoid culture system supports ex vivo expansion of tissue-resident stem cells by reconstituting the microenvironment or “niche” essential for stem cell self-renewal using mitogenic stimuli and extracellular matrix (Figure 1A). Although the composition of the stem cell niche varies from organ to organ, the organoid culture of most human GI epithelial tissues commonly requires Wnt activators (Wnt-3A and R-spondin), receptor tyrosine kinase ligands (epidermal growth factor [EGF] and fibroblast growth factor [FGF] 10), a BMP inhibitor (Noggin), and a transforming growth factor (TGF)–β inhibitor, along with additional factors contingent on the tissue origin.35Date S. Sato T. Mini-gut organoids: reconstitution of the stem cell niche.Annu Rev Cell Dev Biol. 2015; 31: 269-289Crossref PubMed Scopus (66) Google Scholar, 36Sato T. Clevers H. SnapShot: growing organoids from stem cells.Cell. 2015; 161: 1700-1700 e1Abstract Full Text PDF PubMed Google Scholar With continuous efforts to tailor the niche factor combination to previously uncultured tissues, the organoid system now encompasses most human GI tissues from the esophagus to large intestine (summarized in Figure 1B). Importantly, organoid culture conditions optimized for healthy tissues in most cases also enable the expansion of diseased epithelium. General protocols for adult stem cell organoid culture selectively expand epithelial cells, and thus patient-derived organoids allows one to investigate epithelium-inherent pathology of GI diseases on an individual patient basis. As alternative strategies to study interactions of GI epithelia with non-epithelial compartment, culture methods involving stromal cells37Ootani A. Li X. Sangiorgi E. et al.Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.Nat Med. 2009; 15: 701-706Crossref PubMed Scopus (409) Google Scholar, 38Wang X. Yamamoto Y. Wilson L.H. et al.Cloning and variation of ground state intestinal stem cells.Nature. 2015; 522: 173-178Crossref PubMed Google Scholar, 39Kabiri Z. Greicius G. Madan B. et al.Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts.Development. 2014; 141: 2206-2215Crossref PubMed Scopus (145) Google Scholar, 40Ohlund D. Handly-Santana A. Biffi G. et al.Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer.J Exp Med. 2017; 214: 579-596Crossref PubMed Scopus (261) Google Scholar, 41Seino T. Kawasaki S. Shimokawa M. et al.Human pancreatic tumor organoids reveal loss of stem cell niche factor dependence during disease progression.Cell Stem Cell. 2018; 22: 454-467 e6Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar or immune cells,42Nozaki K. Mochizuki W. Matsumoto Y. et al.Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes.J Gastroenterol. 2016; 51: 206-213Crossref PubMed Google Scholar, 43Dijkstra K.K. Cattaneo C.M. Weeber F. et al.Generation of Tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids.Cell. 2018; 174: 1586-1598 e12Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar as well as pluripotent stem cell-derived organoids that are typically coated with stromal cells, can be exploited. The archetype CRISPR-Cas9 system for mammalian genome editing consists of Cas9 nuclease derived from Streptococcus pyogenes and custom guide RNA that recognizes and targets a specified DNA sequence preceding the protospacer adjacent motif sequence. Although the requirement for this motif sequence, which substantially vary between Cas9 variants, restricts the scope of guide RNA design, CRISPR-Cas9 enables generation of a DNA double-strand break at a specific genomic position. Double-strand breaks in mammalian DNA are repaired through two mechanisms, non-homologous end joining (NHEJ) and homology-directed repair (HDR). The error-prone NHEJ randomly inserts indels during the repair, and biallelic introduction of indel mutations leads to gene knockout. HDR, which is normally harnessed to replace a damaged allele using existing intact genome, can be coopted for gene knock-in when customized DNA templates are co-delivered with CRISPR-Cas9. For example, single-strand oligonucleotides or plasmids harboring nucleotide variants and homology arms are used for introducing missense mutations, and HDR templates with functional gene cassettes enable integration of selection markers or gene reporters. While the CRISPR-Cas9 technology has been innovated for a range of purposes, such as DNA base editing; RNA targeting; gene expression regulation; epigenome editing; and the visualization of specific DNA loci,4Komor A.C. Badran A.H. Liu D.R. CRISPR-based technologies for the manipulation of eukaryotic genomes.Cell. 2017; 168: 20-36Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 5Adli M. The CRISPR tool kit for genome editing and beyond.Nat Commun. 2018; 9: 1911Crossref PubMed Scopus (173) Google Scholar the use of CRISPR-Cas9 on organoids has basically adopted NHEJ and HDR mechanisms to engineer genes of interest. Indeed, as the organoid system allows the expansion of non-transformed tissues without losing genetic and phenotypic stability, organoids are excellent tools for analyzing gene functions via prospective genome engineering. Previous studies have used different methods to install CRISPR-Cas9 into organoids, including liposomal transfection, electroporation and viral infection, all of which attained successful genome editing (Figure 2A). Nonetheless, it should be noted that the efficiency of genome editing in GI organoids is highly subject to experimental variables, including the recovery after single-cell dissociation, methods for CRISPR-Cas9 delivery, and the cleavage efficiency of CRISPR-Cas9 itself. Positive selection and enrichment of organoids that have undergone CRISPR-Cas9 delivery and subsequent genome editing is therefore mandatory, or otherwise, labor-intensive organoid cloning, followed by sequencing of expanded organoid clones is required. For example, cancer modeling by CRISPR-Cas9, discussed here in detail, has exploited the fact that specific oncogenic mutations relieve the dependence on individual niche growth factors, thus allowing the enrichment of organoids with desired mutations.41Seino T. Kawasaki S. Shimokawa M. et al.Human pancreatic tumor organoids reveal loss of stem cell niche factor dependence during disease progression.Cell Stem Cell. 2018; 22: 454-467 e6Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 44Matano M. Date S. Shimokawa M. et al.Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.Nat Med. 2015; 21: 256-262Crossref PubMed Scopus (374) Google Scholar, 45Drost J. van Jaarsveld R.H. Ponsioen B. et al.Sequential cancer mutations in cultured human intestinal stem cells.Nature. 2015; 521: 43-47Crossref PubMed Scopus (327) Google Scholar In other situations where such niche-based selection is impractical, organoids of interest can alternatively be retrieved by co-introducing a selection vector with CRISPR-Cas9, or by knocking in a positive selection cassette by HDR.46Fujii M. Matano M. Nanki K. et al.Efficient genetic engineering of human intestinal organoids using electroporation.Nat Protoc. 2015; 10: 1474-1485Crossref PubMed Google Scholar In silico guide RNA design based on on-target activity prediction47Chari R. Mali P. Moosburner M. et al.Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach.Nat Methods. 2015; 12: 823-826Crossref PubMed Scopus (160) Google Scholar, 48Doench J.G. Fusi N. Sullender M. et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.Nat Biotechnol. 2016; 34: 184-191Crossref PubMed Scopus (708) Google Scholar and using multiple guide RNAs per gene may also aid in enhancing genome-editing efficiency in organoids. On another front, off-target cleavage by CRISPR-Cas9 cannot be overlooked especially in gene knockout experiments. Direct survey of potential off-target sites by deep sequencing is costly and lengthy, and efforts have been made to reduce off-target effects, for instance, by selecting highly specific guide RNA sequences,48Doench J.G. Fusi N. Sullender M. et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.Nat Biotechnol. 2016; 34: 184-191Crossref PubMed Scopus (708) Google Scholar, 49Haeussler M. Schonig K. Eckert H. et al.Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.Genome Biol. 2016; 17: 148Crossref PubMed Scopus (344) Google Scholar re-engineering Cas9 nuclease derived from Streptococcus pyogenes,50Kleinstiver B.P. Pattanayak V. Prew M.S. et al.High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.Nature. 2016; 529: 490-495Crossref PubMed Scopus (920) Google Scholar or using paired nickase Cas9.51Ran F.A. Hsu P.D. Lin C.Y. et al.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.Cell. 2013; 154: 1380-1389Abstract Full Text Full Text PDF PubMed Scopus (1674) Google Scholar Reproducing identical phenotypes using independent guide RNAs targeting the same gene may alternatively decrease the odds of off-target effects. A recent report has also demonstrated frequent large genomic deletions and rearrangements occur at CRISPR-Cas9 on-target sites in mouse embryonic stem cells and human cell lines.52Kosicki M. Tomberg K. Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.Nat Biotechnol. 2018; 36: 765-771Crossref PubMed Scopus (28) Google Scholar Although similar phenomena have not been demonstrated in organoids, whether CRISPR-Cas9 has introduced desired mutations at correct positions may thus be exhaustively evaluated. The intestinal epithelium is one of the most rapidly renewing tissues in the mammalian body, and intestinal stem cells located at the crypt bottom essentially fuel this process.53Clevers H. The intestinal crypt, a prototype stem cell compartment.Cell. 2013; 154: 274-284Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar The discovery of Lgr5 as a marker for intestinal stem cells54Barker N. van Es J.H. Kuipers J. et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (2967) Google Scholar has expanded our understanding of the regulatory niche signals that coordinately direct the intestinal stem cells to properly execute their functions. Based on this progress, we established a method for expanding intestinal crypts and stem cells as 3D crypt-villus structures, that we then named “organoids.”7Sato T. Vries R.G. Snippert H.J. et al.Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (2436) Google Scholar The organoid culture of human intestinal stem cells requires a defined growth factor combination consisting of Wnt-3A, R-spondin, EGF, Noggin, a TGF-β inhibitor, and a p38 MAPK inhibitor.9Sato T. Stange D.E. Ferrante M. et al.Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (1053) Google Scholar This organoid expansion condition enriches human intestinal organoids with undifferentiated cells; subsequent withdrawal of Wnt activators and the p38 inhibitor is necessary to gear the organoid cells toward terminal differentiation. Nonetheless, this technology was the first to allow long-term 3D expansion of human stem cells of endodermal organs; this achievement has paved the way for the later endeavors to establish culture systems for other human GI tissues. The ability of the organoids to directly reconstitute human tissue ex vivo facilitated the characterization of diseased epithelium-derived organoids and their usage for various applications. The first monogenic disease to be modeled using patient-derived organoids was cystic fibrosis (CF), a debilitating hereditary disorder caused by mutations in the CFTR gene encoding an ATP-gated chloride channel.55Elborn J.S. Cystic fibrosis.Lancet. 2016; 388: 2519-2531Abstract Full Text Full Text PDF PubMed Google Scholar To examine the phenotypes of CF organoids, Dekkers et al56Dekkers J.F. Wiegerinck C.L. de Jonge H.R. et al.A functional CFTR assay using primary cystic fibrosis intestinal organoids.Nat Med. 2013; 19: 939-945Crossref PubMed Scopus (371) Google Scholar generated rectal organoids from endoscopic biopsy samples of CF patients. While healthy intestinal organoids exhibited a swelling response to a treatment with forskolin, resulting from fluid secretion into the organoid lumen upon CFTR opening, CF organoids showed moderate to no swelling, reflecting their defective CFTR function. CF organoids also reproduced genotype-specific response to CFTR modulators and correctors, which are currently approved therapeutic agents for CF patients with specific CFTR mutations. The identification of potential responders to these drugs has been challenging due to uncommon CFTR mutations present in nearly half of CF subjects. The facile and reproducible organoid swelling assay may therefore afford a useful option for optimizing the treatment strategy for CF patients.57Dekkers J.F. Berkers G. Kruisselbrink E. et al.Characterizing responses to CFTR-modulating drugs using rectal organoids derived from subjects with cystic fibrosis.Sci Transl Med. 2016; 8: 344ra84Crossref PubMed Scopus (156) Google Scholar Indeed, the registration of the Vertex (Haarlem, The Netherlands) drug Orkambi in the Netherlands now includes any CF patient with a positive organoid response, independent of the type of CFTR mutation. To physiologically restore CFTR function in CF patient–derived intestinal organoids, Schwank et al58Schwank G. Koo B.K. Sasselli V. et al.Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.Cell Stem Cell. 2013; 13: 653-658Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar accurately repaired their defective CFTR gene by using CRISPR-Cas9 and HDR. CFTR-corrected CF organoids regained the swelling response to forskolin treatment with levels comparable to those of wild-type intestinal organoids. These experiments performed in 2013 collectively demonstrated for the first time that specific hereditary disease genes can be edited precisely in patient tissue stem cells by CRISPR-Cas9. Later studies have derived intestinal organoids from patients with other rare Mendelian diseases: multiple intestinal atresia (MIA),59Bigorgne A.E. Farin H.F. Lemoine R. et al.TTC7A mutations disrupt intestinal epithelial apicobasal polarity.J Clin Invest. 2014; 124: 328-337Crossref PubMed Scopus (76) Google Scholar microvillus inclusion disease (MVID),60Wiegerinck C.L. Janecke A.R. Schneeberger K. et al.Loss of syntaxin 3 causes variant microvillus inclusion disease.Gastroenterology. 2014; 147: 65-68 e10Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar and DGAT1 deficiency.61van Rijn J.M. Ardy R.C. Kuloglu Z. et al.Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency.Gastroenterology. 2018; 155: 130-143.e15Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar MIA is characterized by multiple atretic segments in the intestine and associated with severe combined immunodeficiency. Intestines of MIA patients show a disorganized architecture with pseudo-stratified appearance and inverse apicobasal polarity. Genome-wide linkage analysis and exome sequencing identified TTC7A mutations as a candidate cause of this disorder.59Bigorgne A.E. Farin H.F. Lemoine R. et al.TTC7A mutations disrupt intestinal epithelial apicobasal polarity.J Clin Invest. 2014; 124: 328-337Crossref PubMed Scopus (76) Google Scholar Intestinal organoids derived from an MIA patient with a biallelic TTC7A germline mutation exhibited inverted apicobasal polarity, consistent with the histology of MIA intestines. MIA patient–derived organoids further showed the activation of ROCK, a component of integrin-cytoskeleton signal. Indeed, a chemical inhibition of ROCK restored the defective polarity of the MIA organoids, suggesting the negative regulation of ROCK by TTC7A. MVID is characterized by intractable diarrhea and malabsorption predominantly associated with MYO5B mutations.62Muller T. Hess M.W. Schiefermeier N. et al.MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity.Nat Genet. 2008; 40: 1163-1165Crossref PubMed Scopus (205) Google Scholar Wiegerinck et al60Wiegerinck C.L. Janecke A.R. Schneeberger K. et al.Loss of syntaxin 3 causes variant microvillus inclusion disease.Gastroenterology. 2014; 147: 65-68 e10Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar recently identified novel homozygous loss-of-function mutations in the STX3 gene in patients with variant MVID. Intestinal organoids derived from these patients reproduced characteristics of the MVID intestine: the depletion of brush border microvilli and the presence of periodic acid-Schiff–positive subapical vesicles, along with the loss of STX3 protein. DGAT1 deficiency is another rare cause of intractable diarrhea and malabsorption starting early in life.63Haas J.T. Winter H.S. Lim E. et al.DGAT1 mutation is linked to a congenital diarrheal disorder.J Clin Invest. 2012; 122: 4680-4684Crossref PubMed Scopus (84) Google Scholar DGAT1 catalyzes the conversion from diacylglycerol and fatty-acyl CoA to triacylglycerol and its defect causes protein-losing enteropathy and fat intolerance. Intestinal organoids derived from patients with DGAT1 mutations showed reduced lipid droplet formation and increased cell death after oleic acid treatment, which mirrored fat intolerance and the resulting intestinal injury in patients with DGAT1 deficiency.61van Rijn J.M. Ardy R.C. Kuloglu Z. et al.Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency.Gastroenterology. 2