Crispr engineering in organoids for gene repair and disease modelling

Crispr engineering in organoids for gene repair and disease modelling

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ABSTRACT Organoids bridge the gap between 2D cell lines and in vivo studies. With their 3D organization and cellular heterogeneity, adult stem cell-derived organoids closely resemble their


tissue of origin. The development of CRISPR-mediated genome engineering and the recent additions of base and prime editing to the CRISPR toolbox have greatly simplified the generation of


exact, isogenic models for Mendelian diseases. Here, we review recent developments in CRISPR-mediated genome engineering and its application in human adult-stem-cell-derived organoids in the


construction of isogenic disease models. These models allow accurate qualification of the impact of allelic disease variants observed in patients. Furthermore, we discuss the use of


organoids as models for safety and efficacy of CRISPR for gene repair. Although transplantation of repaired tissue remains challenging, benchmarking CRISPR tools in organoids can bring


genome engineering one step closer to patients. KEY POINTS * CRISPR–Cas9-mediated genome engineering acts by introducing double-stranded DNA breaks into the genome. The damage repair process


can be used for gene knockout or precise targeted introduction of exogenous DNA. * Next-generation CRISPR tools, including base and prime editing, allow for induction of precise base


changes and small insertions and deletions, bypassing potentially deleterious double-stranded DNA breaks. * Owing to their 3D organization, adult-stem-cell-derived organoids closely resemble


the tissue of origin and are therefore a good model system to study human health and disease. * CRISPR–Cas9-mediated genome engineering can be used to create isogenic models to investigate


the onset, cause and treatment of human diseases. * CRISPR tools can be benchmarked for efficiency and safety by studying gene repair ex vivo in adult-stem-cell-derived organoids,


facilitating CRISPR–Cas9 clinical translation. * Ex vivo repaired adult-stem-cell-derived organoids can potentially be transplanted into patients to relieve disease phenotypes. Access


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support SIMILAR CONTENT BEING VIEWED BY OTHERS CRISPR SOMATIC GENOME ENGINEERING AND CANCER MODELING IN THE MOUSE PANCREAS AND LIVER Article 14 March 2022 PRIME EDITING FOR FUNCTIONAL


REPAIR IN PATIENT-DERIVED DISEASE MODELS Article Open access 23 October 2020 THE NIH SOMATIC CELL GENOME EDITING PROGRAM Article Open access 07 April 2021 REFERENCES * Visscher, P. M. et al.


10 years of GWAS discovery: biology, function, and translation. _Am. J. Hum. Genet._ 101, 5–22 (2017). Article  Google Scholar  * Xuan, J., Yu, Y., Qing, T., Guo, L. & Shi, L.


Next-generation sequencing in the clinic: promises and challenges. _Cancer Lett._ 340, 284–295 (2013). Article  Google Scholar  * Jinek, M. et al. A programmable dual-RNA-guided DNA


endonuclease in adaptive bacterial immunity. _Science_ 337, 816–821 (2012). THIS ARTICLE CONTAINS THE FIRST DESCRIPTION OF THE CRISPR–CAS9 SYSTEM AS A POTENTIAL TOOL FOR RNA-PROGRAMMABLE


GENOME ENGINEERING. Article  Google Scholar  * Mali, P. et al. RNA-guided human genome engineering via Cas9. _Science_ 339, 823–826 (2013). Article  Google Scholar  * Cong, L. et al.


Multiplex genome engineering using CRISPR/Cas systems. _Science_ 339, 819–823 (2013). Article  Google Scholar  * Kapałczyńska, M. et al. 2D and 3D cell cultures — a comparison of different


types of cancer cell cultures. _Arch. Med. Sci._ 14, 910–919 (2018). Google Scholar  * Clevers, H. Modeling development and disease with organoids. _Cell_ 165, 1586–1597 (2016). Article 


Google Scholar  * Kim, J., Koo, B. K. & Knoblich, J. A. Human organoids: model systems for human biology and medicine. _Nat. Rev. Mol. Cell Biol._ 21, 571–584 (2020). Article  Google


Scholar  * Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. _Gastroenterology_ 141, 1762–1772 (2011). Article


  Google Scholar  * Barker, N. et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. _Cell Stem Cell_ 6, 25–36 (2010). Article  Google


Scholar  * Schutgens, F. et al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. _Nat. Biotechnol._ 37, 303–313 (2019). Article  Google Scholar  * Huch,


M. et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. _EMBO J._ 32, 2708–2721 (2013). Article  Google Scholar  * Linnemann, J. R.


et al. Quantification of regenerative potential in primary human mammary epithelial cells. _Development_ 142, 3239–3251 (2015). Google Scholar  * Boretto, M. et al. Development of organoids


from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability. _Development_ 144, 1775–1786 (2017). Google Scholar  * Lõhmussaar, K. et al.


Patient-derived organoids model cervical tissue dynamics and viral oncogenesis in cervical cancer. _Cell Stem Cell_ 28, 1380–1396.e6 (2021). Article  Google Scholar  * Huch, M. et al. In


vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. _Nature_ 494, 247–250 (2013). Article  Google Scholar  * Hu, H. et al. Long-term expansion of functional


mouse and human hepatocytes as 3D organoids. _Cell_ 175, 1591–1606.e19 (2018). Article  Google Scholar  * Sachs, N. et al. Long‐term expanding human airway organoids for disease modeling.


_EMBO J._ 38, e100300 (2019). Article  Google Scholar  * Nikolić, M. Z. et al. Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term


self-renewing organoids. _eLife_ 6, e26575 (2017). Article  Google Scholar  * Ren, W. et al. Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo.


_Proc. Natl Acad. Sci. USA_ 111, 16401–16406 (2014). Article  Google Scholar  * Bannier-Hélaouët, M. et al. Exploring the human lacrimal gland using organoids and single-cell sequencing.


_Cell Stem Cell_ 28, 1221–1232.e7 (2021). Article  Google Scholar  * Mullenders, J. et al. Mouse and human urothelial cancer organoids: a tool for bladder cancer research. _Proc. Natl Acad.


Sci. USA_ 116, 4567–4574 (2019). Article  Google Scholar  * Karthaus, W. R. et al. Identification of multipotent luminal progenitor cells in human prostate organoid cultures. _Cell_ 159,


163–175 (2014). Article  Google Scholar  * van der Vaart, J. et al. Adult mouse and human organoids derived from thyroid follicular cells and modeling of Graves’ hyperthyroidism. _Proc. Natl


Acad. Sci. USA_ 118, e2117017118 (2021). Article  Google Scholar  * Ogundipe, V. M. L. et al. Generation and differentiation of adult tissue-derived human thyroid organoids. _Stem Cell


Rep._ 16, 913–925 (2021). Article  Google Scholar  * Sato, T. et al. Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. _Nature_ 459, 262–265 (2009).


THIS ARTICLE DESCRIBES THE FIRST ADULT-STEM-CELL-DERIVED ORGANOID CULTURES DERIVED FROM THE MOUSE INTESTINE. Article  Google Scholar  * Wright, A. V., Nuñez, J. K. & Doudna, J. A.


Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. _Cell_ 164, 29–44 (2016). Article  Google Scholar  * Sternberg, S. H., Redding, S., Jinek, M.,


Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. _Nature_ 507, 62–67 (2014). Article  Google Scholar  * Ghezraoui, H. et al. Chromosomal


translocations in human cells are generated by canonical nonhomologous end-joining. _Mol. Cell_ 55, 829–842 (2014). Article  Google Scholar  * Zhang, X. H., Tee, L. Y., Wang, X. G., Huang,


Q. S. & Yang, S. H. Off-target effects in CRISPR/Cas9-mediated genome engineering. _Mol. Ther. Nucleic Acids_ 4, e264 (2015). Article  Google Scholar  * Kleinstiver, B. P. et al.


High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. _Nature_ 529, 490–495 (2016). Article  Google Scholar  * Vakulskas, C. A. et al. A high-fidelity Cas9


mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. _Nat. Med._ 24, 1216–1224 (2018). Article  Google Scholar  *


Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. _Nat. Biotechnol._ 33, 187–197 (2015). Article  Google Scholar  * Tsai, S. Q. et


al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. _Nat. Methods_ 14, 607–614 (2017). Article  Google Scholar  * Kosicki, M., Tomberg, K.


& Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. _Nat. Biotechnol._ 36, 765–771 (2018). Article  Google Scholar  *


Leibowitz, M. L. et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. _Nat. Genet._ 53, 895–905 (2021). Article  Google Scholar  * Branzei, D. & Foiani, M.


Regulation of DNA repair throughout the cell cycle. _Nat. Rev. Mol. Cell Biol._ 9, 297–308 (2008). Article  Google Scholar  * Maruyama, T. et al. Increasing the efficiency of precise genome


editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. _Nat. Biotechnol._ 33, 538–542 (2015). Article  Google Scholar  * Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A.


Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. _eLife_ 3, e04766 (2014). Article  Google Scholar  * Rees, H. A. & Liu, D. R. Base


editing: precision chemistry on the genome and transcriptome of living cells. _Nat. Rev. Genet._ 19, 770–788 (2018). Article  Google Scholar  * Qi, L. S. et al. Repurposing CRISPR as an


RNA-guided platform for sequence-specific control of gene expression. _Cell_ 152, 1173–1183 (2013). Article  Google Scholar  * Chavez, A. et al. Highly-efficient Cas9-mediated


transcriptional programming. _Nat. Methods_ 12, 326–328 (2015). Article  Google Scholar  * Vojta, A. et al. Repurposing the CRISPR-Cas9 system for targeted DNA methylation. _Nucleic Acids


Res._ 44, 5615–5628 (2016). Article  Google Scholar  * Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without


double-stranded DNA cleavage. _Nature_ 533, 420–424 (2016). THIS ARTICLE REPORTS THE FIRST BASE-EDITING SYSTEM BY FUSING CYTIDINE DEAMINASE APOBEC TO NICKASE- AND NUCLEASE-INACTIVE CAS9


ALLOWING FOR C-TO-T BASE EDITING. Article  Google Scholar  * Cascalho, M. Advantages and disadvantages of cytidine deamination. _J. Immunol._ 172, 6513–6518 (2004). Article  Google Scholar 


* Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. _Sci. Adv._ 3,


eaao4774 (2017). Article  Google Scholar  * Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. _Nat. Biotechnol._ 36, 888–896 (2018). Article 


Google Scholar  * Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. _Nat. Biotechnol._ 36, 843–848 (2018). Article 


Google Scholar  * Levy, J. M. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. _Nat. Biomed. Eng._ 4,


97–110 (2020). Article  Google Scholar  * Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. _Science_ 353, aaf8729 (2016).


Article  Google Scholar  * Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. _Nature_ 551, 464–471 (2017). THIS ARTICLE DESCRIBES THE FIRST


ADENINE BASE EDITOR THAT ALLOWS FOR A-TO-G BASE EDITING WITHOUT THE NEED FOR DSBS. Article  Google Scholar  * Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous


directed evolution of biomolecules. _Nature_ 472, 499–503 (2011). Article  Google Scholar  * Gaudelli, N. M. et al. Directed evolution of adenine base editors with increased activity and


therapeutic application. _Nat. Biotechnol._ 38, 892–900 (2020). Article  Google Scholar  * Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain


compatibility and activity. _Nat. Biotechnol._ 38, 883–891 (2020). Article  Google Scholar  * Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.


_Nature_ 556, 57–63 (2018). Article  Google Scholar  * Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. _Science._ 9, 1259–1262 (2018). Article  Google


Scholar  * Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR–Cas9 variants. _Science._ 368, 290–296


(2020). Article  Google Scholar  * Yu, S.-Y. et al. Increasing the targeting scope of CRISPR base editing system beyond NGG. _CRISPR J._ 5, 187–202 (2022). Article  Google Scholar  *


Pavlov, Y. I. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. _Science_ 8, 647–656 (2019). Google Scholar  * Zuo, E. et al. Cytosine base


editor generates substantial off-target single-nucleotide variants in mouse embryos. _Science_ 292, eaav9973 (2019). Google Scholar  * Yu, Y. et al. Cytosine base editors with minimized


unguided DNA and RNA off-target events and high on-target activity. _Nat. Commun._ 11, 2052 (2020). Article  Google Scholar  * Kurt, I. C. et al. CRISPR C-to-G base editors for inducing


targeted DNA transversions in human cells. _Nat. Biotechnol._ 39, 41–46 (2021). Article  Google Scholar  * Koblan, L. W. et al. Efficient C•G-to-G•C base editors developed using CRISPRi


screens, target-library analysis, and machine learning. _Nat. Biotechnol._ 39, 1414–1425 (2021). Article  Google Scholar  * Anzalone, A. V. et al. Search-and-replace genome editing without


double-strand breaks or donor DNA. _Nature_ 576, 149–157 (2019). THIS ARTICLE PRESENTS PRIME EDITING AS A TOOL THAT CAN POTENTIALLY REPAIR 89% OF ALL DISEASE-CAUSING MUTATIONS OBSERVED IN


HUMANS WITHOUT THE NEED FOR DSBS. Article  Google Scholar  * Alexandrov, L. B. et al. Signatures of mutational processes in human cancer. _Nature_ 500, 415–421 (2013). Article  Google


Scholar  * Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. _Nat. Biotechnol._ 40, 731–740 (2021). Article


  Google Scholar  * Lin, Q. et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. _Nat. Biotechnol._ 39, 923–927 (2021). Article  Google Scholar  * Choi, J. et al.


Precise genomic deletions using paired prime editing. _Nat. Biotechnol._ 40, 218–226 (2022). Article  Google Scholar  * Nelson, J. W. et al. Engineered pegRNAs improve prime editing


efficiency. _Nat. Biotechnol._ https://doi.org/10.1038/s41587-021-01039-7 (2021). Article  Google Scholar  * Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular


determinants of editing outcomes. _Cell_ 184, 5635–5652.e29 (2021). Article  Google Scholar  * Fearon, E. F. & Vogelstein, B. A genetic model for colorectal tumorigenesis. _Cell_ 61,


759–767 (1990). Article  Google Scholar  * Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. _Nature_ 521, 43–47 (2015). Article  Google Scholar  *


Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids. _Nat. Med._ 21, 256–262 (2015). Article  Google Scholar  * Dekkers, J. F.


et al. Modeling breast cancer using CRISPR-Cas9-mediated engineering of human breast organoids. _J. Natl. Cancer Inst._ 112, 540–544 (2020). Article  Google Scholar  * Artegiani, B. et al.


Probing the tumor suppressor function of BAP1 in CRISPR-engineered human liver organoids. _Cell Stem Cell_ 24, 927–943.e6 (2019). Article  Google Scholar  * Seino, T. et al. Human pancreatic


tumor organoids reveal loss of stem cell niche factor dependence during disease progression. _Cell Stem Cell_ 22, 454–467.e6 (2018). Article  Google Scholar  * Lee, J. et al. Reconstituting


development of pancreatic intraepithelial neoplasia from primary human pancreas duct cells. _Nat. Commun._ 8, 14686 (2017). Article  Google Scholar  * Drost, J. et al. Use of


CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer. _Science_ 358, 234–238 (2017). Article  Google Scholar  * Jager, M. et al. Deficiency of


nucleotide excision repair is associated with mutational signature observed in cancer. _Genome Res._ 29, 1067–1077 (2019). Article  Google Scholar  * Kawasaki, K. et al. Chromosome


engineering of human colon-derived organoids to develop a model of traditional serrated adenoma. _Gastroenterology_ 158, 638–651.e8 (2020). Article  Google Scholar  * Artegiani, B. et al.


Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing. _Nat. Cell Biol._ 22, 321–331 (2020). Article  Google Scholar  *


Lo, Y. H. et al. A CRISPR/Cas9-engineered _ARID1A_-deficient human gastric cancer organoid model reveals essential and nonessential modes of oncogenic transformation. _Cancer Discov._ 11,


1562–1581 (2021). Article  Google Scholar  * Kawasaki, K. et al. An organoid biobank of neuroendocrine neoplasms enables genotype–phenotype mapping. _Cell_ 183, 1420–1435.e21 (2020). Article


  Google Scholar  * Yan, H. H. N. et al. Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles. _Gut_ 69, 2165–2179 (2020). Article  Google Scholar  *


Post, J. B. et al. CRISPR-induced RASGAP deficiencies in colorectal cancer organoids reveal that only loss of NF1 promotes resistance to EGFR inhibition. _Oncotarget_ 10, 1440–1457 (2019).


Article  Google Scholar  * Bock, C. et al. High-content CRISPR screening. _Nat. Rev. Methods Prim._ 2, 8 (2022). Article  Google Scholar  * Wang, T. et al. Identification and


characterization of essential genes in the human genome. _Science_ 350, 1096–1101 (2015). Article  Google Scholar  * Shalem, O. et al. Genome-scale CRISPR–Cas9 knockout screening in human


cells. _Science_ 343, 84–88 (2014). Article  Google Scholar  * Michels, B. E. et al. Pooled in vitro and in vivo CRISPR–Cas9 screening identifies tumor suppressors in human colon organoids.


_Cell Stem Cell_ 26, 782–792.e7 (2020). Article  Google Scholar  * Ringel, T. et al. Genome-scale CRISPR screening in human intestinal organoids identifies drivers of TGF-β resistance. _Cell


Stem Cell_ 26, 431–440.e8 (2020). Article  Google Scholar  * Boettcher, S. et al. A dominant-negative effect drives selection of TP53 missense mutations in myeloid malignancies. _Science_


365, 599–604 (2019). Article  Google Scholar  * Stolze, B., Reinhart, S., Bulllinger, L., Fröhling, S. & Scholl, C. Comparative analysis of KRAS codon 12, 13, 18, 61, and 117 mutations


using human MCF10A isogenic cell lines. _Sci. Rep._ 5, 8535 (2014). Article  Google Scholar  * Geurts, M. H. et al. Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair


in organoids. _Life Sci. Alliance_ 4, 1–12 (2021). Article  Google Scholar  * Schene, I. F. et al. Prime editing for functional repair in patient-derived disease models. _Nat. Commun._ 11,


5352 (2020). Article  Google Scholar  * van Rijn, J. M. et al. Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency. _Gastroenterology_ 155, 130–143.e15 (2018).


Article  Google Scholar  * Nanki, K. et al. Somatic inflammatory gene mutations in human ulcerative colitis epithelium. _Nature_ 577, 254–259 (2020). Article  Google Scholar  * Lamers, M.


M. et al. SARS-CoV-2 productively infects human gut enterocytes. _Science_ 369, 50–54 (2020). Article  Google Scholar  * Zhou, J. et al. Infection of bat and human intestinal organoids by


SARS-CoV-2. _Nat. Med._ 26, 1077–1083 (2020). Article  Google Scholar  * Geurts, M. H., van der Vaart, J., Beumer, J. & Clevers, H. The organoid platform: promises and challenges as


tools in the fight against COVID-19. _Stem Cell Rep._ 16, 412–418 (2021). Article  Google Scholar  * Beumer, J. et al. A CRISPR/Cas9 genetically engineered organoid biobank reveals essential


host factors for coronaviruses. _Nat. Commun._ 12, 5498 (2021). Article  Google Scholar  * Veres, A. et al. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN


targeted human stem cell clones detected by whole-genome sequencing. _Cell Stem Cell_ 15, 27–30 (2014). Article  Google Scholar  * Wu, X. et al. Genome-wide binding of the CRISPR


endonuclease Cas9 in mammalian cells. _Nat. Biotechnol._ 32, 670–676 (2014). Article  Google Scholar  * Lombaert, I. M. A. et al. Rescue of salivary gland function after stem cell


transplantation in irradiated glands. _PLoS One_ 3, e2063 (2008). Article  Google Scholar  * Schwank, G. et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of


cystic fibrosis patients. _Cell Stem Cell_ 13, 653–658 (2013). THIS ARTICLE REPORTS THE FIRST PROOF OF THE POTENTIAL CLINICAL APPLICATION OF CRISPR BY REPAIRING THE MOST COMMON MUTATION THAT


CAUSES CYSTIC FIBROSIS IN PATIENT-DERIVED INTESTINAL ORGANOIDS. Article  Google Scholar  * Sosnay, P. R. et al. Defining the disease liability of variants in the cystic fibrosis


transmembrane conductance regulator gene. _Nat. Genet._ 45, 1160–1167 (2013). Article  Google Scholar  * Dekkers, J. F. et al. A functional CFTR assay using primary cystic fibrosis


intestinal organoids. _Nat. Med._ 19, 939–945 (2013). Article  Google Scholar  * Dekkers, J. F. et al. Characterizing responses to CFTR-modulating drugs using rectal organoids derived from


subjects with cystic fibrosis. _Sci. Transl. Med._ 8, 344ra84–344ra84 (2016). Article  Google Scholar  * Berkers, G. et al. Rectal organoids enable personalized treatment of cystic fibrosis.


_Cell Rep._ 26, 1701–1708.e3 (2019). Article  Google Scholar  * Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. _Cell_ 163, 759–771 (2015).


Article  Google Scholar  * Maule, G. et al. Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing. _Nat. Commun._ 10, 3556 (2019). Article  Google


Scholar  * Geurts, M. H. et al. CRISPR-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank. _Cell Stem Cell_ 26, 503–510.e7 (2020). THIS ARTICLE REPORTS


THE FIRST PROOF OF DSB-FREE GENE REPAIR IN ADULT-STEM-CELL-DERIVED ORGANOIDS BY REPAIRING MUTATIONS THAT CAUSE CYSTIC FIBROSIS IN PATIENT-DERIVED ORGANOIDS WITHOUT GENOME-WIDE OFF-TARGET


EFFECTS. Article  Google Scholar  * Schene, I. F. et al. Mutation-specific reporter for optimization and enrichment of prime editing. _Nat. Commun._ 13, 1028 (2022). Article  Google Scholar


  * van der Vaart, J. et al. Modelling of primary ciliary dyskinesia using patient‐derived airway organoids. _EMBO Rep._ 22, e52058 (2021). Google Scholar  * Kuscu, C. et al. CRISPR-STOP:


gene silencing through base-editing-induced nonsense mutations. _Nat. Methods_ 14, 710–712 (2017). Article  Google Scholar  * Wang, X. et al. Efficient gene silencing by adenine base


editor-mediated start codon mutation. _Mol. Ther._ 28, 431–440 (2020). Article  Google Scholar  * Kluesner, M. G. et al. CRISPR–Cas9 cytidine and adenosine base editing of splice-sites


mediates highly-efficient disruption of proteins in primary and immortalized cells. _Nat. Commun._ 12, 2437 (2021). Article  Google Scholar  * Conant, D. et al. Inference of CRISPR edits


from sanger trace data. _CRISPR J._ 5, 123–130 (2022). Article  Google Scholar  * Arbab, M. et al. Determinants of base editing outcomes from target library analysis and machine learning.


_Cell_ 182, 463–480.e30 (2020). Article  Google Scholar  * Andersson-Rolf, A. et al. One-step generation of conditional and reversible gene knockouts. _Nat. Methods_ 14, 287–289 (2017).


Article  Google Scholar  * Sun, D. et al. A functional genetic toolbox for human tissue-derived organoids. _eLife_ 10, e67886 (2021). Article  Google Scholar  * Yarnall, M. T. N. et al.


Drag-and-drop genome insertion of large sequences without DNA cleavage using CRISPR-directed integrases. _Nat. Biotechnol._ https://doi.org/10.1038/s41587-022-01527-4 (2022). * Price, S. et


al. A suspension technique for efficient large-scale cancer organoid culturing and perturbation screens. _Sci. Rep._ 12, 5571 (2022). Article  Google Scholar  * Hanna, R. E. et al. Massively


parallel assessment of human variants with base editor screens. _Cell_ 184, 1064–1080.e20 (2021). Article  Google Scholar  * Drost, J. & Clevers, H. Translational applications of adult


stem cell-derived organoids. _Development_ 144, 968–975 (2017). Article  Google Scholar  * Yui, S. et al. Functional engraftment of colon epithelium expanded in vitro from a single adult


Lgr5+ stem cell. _Nat. Med._ 18, 618–623 (2012). Article  Google Scholar  * Pringle, S. et al. Human salivary gland stem cells functionally restore radiation damaged salivary glands. _Stem


Cell_ 34, 640–652 (2016). Article  Google Scholar  * Sampaziotis, F. et al. Cholangiocyte organoids can repair bile ducts after transplantation in the human liver. _Science_ 371, 839–846


(2021). Article  Google Scholar  * Gillmore, J. D. et al. CRISPR–Cas9 in vivo gene editing for transthyretin amyloidosis. _N. Engl. J. Med._ 385, 493–502 (2021). THIS ARTICLE DESCRIBES A


LANDMARK CLINICAL TRIAL IN WHICH PATIENTS ARE INJECTED WITH NUCLEASE-ACTIVE CAS9 AND A SGRNA TARGETING THE TRANSTHYRETIN GENE THAT CAUSES AMYLOID PLAQUES IN THE LIVER. Article  Google


Scholar  * Doman, J. L., Raguram, A., Newby, G. A. & Liu, D. R. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. _Nat. Biotechnol._ 38,


620–628 (2020). Article  Google Scholar  * Aida, T. et al. Prime editing primarily induces undesired outcomes in mice. Preprint at _bioRxiv_


https://www.biorxiv.org/content/10.1101/2020.08.06.239723v1 (2020). * Shen, B. et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. _Nat. Methods_ 11,


399–402 (2014). Article  Google Scholar  * Muller, H. J. Artificial transmutation of the gene. _Science_ 66, 84–87 (1927). Article  Google Scholar  * Brenner, S. The genetics of


_Ceanorhabditis elegans_. _Genetics_ 77, 71–94 (1974). Article  Google Scholar  * Nüsslein-volhard, C. & Wieschaus, E. Mutations affecting segment number and polarity in _Drosophila_.


_Nature_ 287, 795–801 (1980). Article  Google Scholar  * Doudna, J. A. & Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. _Science_ 346, 1258096–1258096 (2014).


Article  Google Scholar  * Scherer, S. & Davis, R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. _Proc. Natl Acad. Sci. USA_ 76, 4951–4955


(1979). Article  Google Scholar  * Smithies, O., Gregg, R. G., Boggst, S. S., Koralewski, M. A. & Kucherlapati, R. S. Insertion of DNA sequences into the human chromosomal β-globin locus


by homologous recombination. _Nature_ 317, 230–236 (1985). Article  Google Scholar  * Rudin, N., Sugarman, E. & Haber, J. E. Genetic and physical analysis of double-strand break repair


and recombination in _Saccharomyces cerevisiae_. _Genetics_ 122, 519–534 (1989). Article  Google Scholar  * Rouet, P., Smih, F. & Jasin, M. Introduction of double-strand breaks into the


genome of mouse cells by expression of a rare-cutting endonuclease. _Mol. Cell. Biol._ 14, 8096–8106 (1994). Google Scholar  * Epinat, J. C. et al. A novel engineered meganuclease induces


homologous recombination in yeast and mammalian cells. _Nucleic Acids Res._ 31, 2952–2962 (2003). Article  Google Scholar  * Wood, A. J. et al. Targeted genome editing across species using


ZFNs and TALENs. _Science_ 333, 307 (2011). Article  Google Scholar  * Hu, J. H., Davis, K. M. & Liu, D. R. Chemical biology approaches to genome editing: understanding, controlling, and


delivering programmable nucleases. _Cell Chem. Biol._ 23, 57–73 (2016). Article  Google Scholar  * Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes.


_Science_ 315, 1709–1712 (2007). Article  Google Scholar  * Brouns, S. J. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. _Science_ 321, 960–965 (2008). Article  Google


Scholar  * Akcakaya, P. et al. In vivo CRISPR editing with no detectable genome-wide off-target mutations. _Nature_ 561, 416–419 (2018). Article  Google Scholar  * Shirley, J. L., de Jong,


Y. P., Terhorst, C. & Herzog, R. W. Immune responses to viral gene therapy vectors. _Mol. Ther._ 28, 709–722 (2020). Article  Google Scholar  * Wu, Z., Asokan, A. & Samulski, R. J.


Adeno-associated virus serotypes: vector toolkit for human gene therapy. _Mol. Ther._ 14, 316–327 (2006). Article  Google Scholar  * Nieuwenhuis, B. et al. Optimization of adeno-associated


viral vector-mediated transduction of the corticospinal tract: comparison of four promoters. _Gene Ther._ 28, 56–74 (2021). Article  Google Scholar  * Burger, C. et al. Recombinant AAV viral


vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. _Mol.


Ther._ 10, 302–317 (2004). Article  Google Scholar  * Naso, M. F., Tomkowicz, B., Perry, W. L. & Strohl, W. R. Adeno-associated virus (AAV) as a vector for gene therapy. _BioDrugs_ 31,


317–334 (2017). Article  Google Scholar  * Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. _Nat. Commun._ 12, 2121 (2021).


Article  Google Scholar  * Böck, D. et al. In vivo prime editing of a metabolic liver disease in mice. _Sci. Transl. Med._ 14, eabl9238 (2022). Article  Google Scholar  * Segel M. et al.


Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for mRNA delivery. _Science_ 185, 882–889 (2021). Article  Google Scholar  * June, C. H., O’Connor, R.


S., Kawalekar, O. U., Ghassemi, S. & Milone, M. C. CAR T cell immunotherapy for human cancer. _Science._ 359, 1361–1365 (2018). Article  Google Scholar  * Frangoul, H. et al. CRISPR–Cas9


gene editing for sickle cell disease and β-thalassemia. _N. Engl. J. Med._ 384, 252–260 (2021). Article  Google Scholar  * Watanabe, S. et al. Transplantation of intestinal organoids into a


mouse model of colitis. _Nat. Protoc._ 17, 649–671 (2022). Article  Google Scholar  * Sugimoto, S. et al. An organoid-based organ-repurposing approach to treat short bowel syndrome.


_Nature_ 592, 99–104 (2021). Google Scholar  Download references ACKNOWLEDGEMENTS The authors thank J. Beumer for providing confocal images of human intestinal organoids, S. Gandhi for


providing confocal images of human fetal hepatocyte organoids and J. van der Vaart for providing confocal images of murine thyroid organoids. AUTHOR INFORMATION Author notes * Maarten H.


Geurts Present address: Xilis BV, Utrecht, The Netherlands * Hans Clevers Present address: Pharma Research Early Development, Roche, Basel, Switzerland AUTHORS AND AFFILIATIONS * Hubrecht


Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center Utrecht, Utrecht, The Netherlands Maarten H. Geurts & Hans Clevers * Oncode Institute,


Hubrecht Institute, Utrecht, The Netherlands Maarten H. Geurts & Hans Clevers Authors * Maarten H. Geurts View author publications You can also search for this author inPubMed Google


Scholar * Hans Clevers View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Hans Clevers. ETHICS DECLARATIONS COMPETING


INTERESTS H.C. is inventor on several patents related to organoid technology; his full disclosure is given at https://www.uu.nl/staff/JCClevers/. H.C. is currently head of pharma Research


Early Development (pRED) at Roche. H.C. holds several patents on organoid technology. Their application numbers, followed by their publication numbers (if applicable), are as follows:


PCT/NL2008/050543, WO2009/022907; PCT/NL2010/000017, WO2010/090513; PCT/IB2011/002167, WO2012/014076; PCT/IB2012/052950, WO2012/168930; PCT/EP2015/060815, WO2015/173425; PCT/EP2015/077990,


WO2016/083613; PCT/EP2015/077988, WO2016/083612; PCT/EP2017/054797, WO2017/149025; PCT/EP2017/065101, WO2017/220586; PCT/EP2018/086716, n/a; and GB1819224.5, n/a. M.H.G. is currently a


scientist at Xilis BV. PEER REVIEW PEER REVIEW INFORMATION _Nature Reviews Bioengineering_ thanks Nicholas Zachos and the other, anonymous, reviewer(s) for their contribution to the peer


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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Geurts, M.H., Clevers, H. CRISPR engineering in organoids for gene repair and disease modelling. _Nat Rev Bioeng_ 1, 32–45 (2023).


https://doi.org/10.1038/s44222-022-00013-5 Download citation * Accepted: 17 November 2022 * Published: 19 January 2023 * Issue Date: January 2023 * DOI:


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