RT Journal Article
SR Electronic
T1 A functional genetic toolbox for human tissue-derived organoids
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.05.04.076067
DO 10.1101/2020.05.04.076067
A1 Sun, Dawei
A1 Evans, Lewis
A1 Lim, Kyungtae
A1 Rawlins, Emma L.
YR 2021
UL http://biorxiv.org/content/early/2021/02/24/2020.05.04.076067.abstract
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.