PT  - JOURNAL ARTICLE
AU  - Sun, Dawei
AU  - Evans, Lewis
AU  - Lim, Kyungtae
AU  - Rawlins, Emma L.
TI  - A functional genetic toolbox for human tissue-derived organoids
AID  - 10.1101/2020.05.04.076067
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2020.05.04.076067
4099  - http://biorxiv.org/content/early/2021/02/24/2020.05.04.076067.short
4100  - http://biorxiv.org/content/early/2021/02/24/2020.05.04.076067.full
AB  - 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. We have systematically developed and optimised a complete genetic toolbox for 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 knock-down, or overexpression, using tightly-inducible CRISPR interference and CRISPR activation; the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids providing a functional counter-part to many on-going descriptive studies, such as the Human Cell Atlas Project.Competing Interest StatementThe authors have declared no competing interest.