A functional genetic toolbox for human tissue-derived organoids

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Perrone, Francesca 
Sokleva, Vanesa 

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.


Funder: Alzheimers Research UK Stem Cell Research Centre

Research Advance, Developmental Biology, organoids, lung, CRISPR-Cas9, homologous gene targeting, inducible CRISPRi, CRISPRa, Human
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eLife Sciences Publications, Ltd
Medical Research Council (MR/P009581/1)
Wellcome Trust (PhD Studentship 109146/Z/15/Z)
National Research Foundation of Korea (2018R1A6A3A03012122)
Wellcome Trust (Core Support for Gurdon Institute 203144/Z/16/Z)
Cancer Research UK (Core Support for Gurdon Institute C6946/A24843)
Medical Research Council (New Investigator Research Grant MR/T001917/1)
Wellcome Trust (PhD studentship 102175/B/13/Z)