A lineage tracing method in organoids

Brain organoids are an important tool in the kit of neuroscientists and neurodevelopmental biologists. Being able to grow a 3D structure in culture, resembling the early stages of the human brain development, has opened research avenues that were previously impractical.

Thanks to the work of Christopher Esk and colleagues (Esk et al.,Science 2020; 370:935-941), it is now possible to perform gene editing of multiple genes in hESCs (human embryonic stem cells) and do lineage tracing in organoids. This approach is called CRISPR–lineage tracing at cellular resolution in heterogeneous tissue (CRISPR-LICHT). Here is a brief overview of how the system works.

The authors generated a hESCs line expressing eCas9-1.1, which has reduced off target expression, and the dTomato reporter; both genes are downstream of a Lox-STOP-Lox cassette. Additionally, a lentivirus carries the gene of the Cre recombinase and the guide RNAs (gRNAs), to complete the TMX-inducible system. Infecting the eCas9 hESCs line with a library of lentiviruses carrying different gRNAs leads to loss of function (LOF) of various target genes. These hESCs can then be used to generate organoids, thus mimicking the effects of the LOF of the genes of interest in the early development of the human brain.

The authors sought to develop a lineage tracing method that allows them to identify differences in lineage growth in organoids. However, the size of different lineages varies throughout the development of the organoid. This makes it difficult to compare different lineages because smaller ones might go undetected. To overcome this hurdle, they developed a two-step barcoding system:

Lineage barcoding: Lentiviruses that each encode a unique lineage barcode and a gRNA → to distinguish different lineages expressing a specific gRNA.

Cell barcoding: Viral barcode inserts are amplified using lineage tracing genomic DNA primers, which also introduces sequencing adaptors. This PCR amplification step uniquely barcodes individual genomically integrated templates corresponding to single cells within a lineage → to determine the cell number within a lineage.

This system yields an increased sensitivity in small lineages, and cell barcodes identify the exact cell numbers in smaller lineages.

After testing the CRISPR-LICHT system, Esk and colleagues applied it to study microcephaly. They screened 172 gene candidates involved in microcephaly in brain organoids, validating, and then identifying 25 of them as not involved in the most common and characterised pathways in microcephaly. They further studied in detail the effect of the LOF of one of these genes in brain organoids.

This system allows a researcher to follow the lineage growth of different cells in organoids, including those leading to smaller sizes and are usually difficult to detect. Furthermore, it is possible to have multiple gRNAs for a single target gene where each of them is coupled to a different lineage barcode. This makes it possible to test and validate various gRNAs, thus enhancing the screening potential.

Written by Chiara Galante; Edited by John JJ Fung. Featured Images: NGC/Design.

Resources:
https://science.sciencemag.org/content/370/6519/935/tab-pdf
https://www.nature.com/articles/d41586-020-03636-z