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New CRISPR tool to visualise cells in human organoids

9 March 2020
Appeared in BioNews 1038

A novel genome editing method may surpass the speed and efficiency of current CRISPR approaches.

CRISPR-HOT (CRISPR/Cas9-mediated homology-independent organoid transgenesis), was developed by researchers at the Hubrecht Institute in the Netherlands, to edit the genomes of organoids: mini-organs, which are grown in the lab from human tissue. The ability to use genome editing in organoids could help in studying biological processes and in modelling diseases.

'Classical CRISPR/Cas9 approaches generate cuts in the genome with the purpose of introducing small mutations or inserting big portions of DNA by exploiting one of [two] mechanisms used by the cells to repair these breaks. However, this last process is extremely inefficient,' said Benedetta Artegiani, co-first author of the study. 'CRISPR-HOT solved this low-efficiency limitation by instead using the other mechanism of DNA repair that was previously thought not to be very feasible'.

Both standard CRISPR and CRISPR-HOT cut through both strands of DNA at a specific location in the genome. Such cuts can be repaired either by joining together the two broken ends or by inserting a small piece of externally provided DNA into the join site.

CRISPR-HOT provided this external DNA in a format that encourages a DNA repair pathway called non-homologous end joining (NHEJ). In contrast, most CRISPR methods rely on the alternative DNA repair pathway, homology-directed repair (HDR). 

'CRISPR-HOT turned out to be more efficient than the original approach to make these knock-in organoids,' said Dr Delilah Hendriks, the other co-first author.

It remains to be seen whether other research groups can reach the same levels of genome editing efficiency as seen in this study, and whether it works equally well in all tissue and organoid types.

CRISPR-HOT was used to edit the genome of organoids grown from adult stem cells. The scientists used CRISPR-HOT to investigate specific genes in the organoids by inserting fluorescent labels into the DNA. 

The fluorescent labels allowed researchers to visualise how hepatocyte liver cells divide in the organoid tissue, and how abnormal cells arise. When they disabled the cancer gene TP53, the unstructured divisions of abnormal hepatocytes were more frequent. This increased their understanding of how abnormal cell divisions may lead to liver cancer.

'We hope that our study contributes to answering complex questions in developmental biology and cancer biology which will be facilitated by visualising genes through CRISPR-HOT,' said Dr Hendriks.

Their results were published in Nature Cell Biology.

CRISPR-HOT: A new tool to 'colour' specific genes and cells
Science Daily |  2 March 2020
CRISPR-HOT: A new tool to visualise cells
Advanced Science News |  4 March 2020
Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing
Nature Cell Biology |  2 March 2020
'HOT' CRISPR tool creates fast and efficient knock-ins
Genetic Engineering and Biotechnology News |  3 March 2020
New CRISPR tool to light up organoid genes
Clinical Omics |  3 March 2020
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