A functional genetic toolbox for human tissue-derived organoids

eLife

Lewis Evans¹  Matthias Zilbauer²  Vanesa Sokleva²  Francesca Perrone²  Emma L Rawlins²  Dawei Sun³  Kyungtae Lim³  Saba Rezakhani⁴  Matthias Lutolf⁴ 

[1] Developmental Biology and Cancer Development, University College London, London, United Kingdom;University of Cambridge, Cambridge, United Kingdom;Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom;École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland;

关键词: organoids;    lung;    CRISPR-Cas9;    homologous gene targeting;    inducible CRISPRi;    CRISPRa;    Human;   

DOI  :  10.7554/eLife.67886

来源: eLife Sciences Publications, Ltd

【 摘 要 】

Human organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact of extrinsic niche factors on stem cells. However, they are rarely used to study endogenous gene function due to the lack of efficient gene manipulation tools. Previously, we established a human foetal lung organoid system (Nikolić et al., 2017). Here, using this organoid system as an example, we have systematically developed and optimised a complete genetic toolbox for use in tissue-derived organoids. This includes ‘Organoid Easytag’, our efficient workflow for targeting all types of gene loci through CRISPR-mediated homologous recombination followed by flow cytometry for enriching correctly targeted cells. Our toolbox also incorporates conditional gene knockdown or overexpression using tightly inducible CRISPR interference and CRISPR activation which is the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids facilitating human disease modelling and providing a functional counterpart to many ongoing descriptive studies, such as the Human Cell Atlas Project.

【 授权许可】

CC BY