Lineage tracing techniques provide information about the history of individual cells, revealing differentiation pathways and progenitor pools. Several lineage tracing methods have been developed, such as those based on fluorescent proteins1, transposons2 or on the insertion of stable DNA barcodes3, but they have various limitations in their ability to reconstruct developmental hierarchies. To address this shortcoming, McKenna et al.4 applied CRISPR–Cas9 technology to develop a DNA barcode system that can generate thousands of unique barcodes as cells divide. In this way, ancestral lineage information is recorded in single cells. The authors applied the method, called genome editing of synthetic target arrays for lineage tracing (GESTALT), to zebrafish development, but in principle it could be adapted to study diverse physiological processes and diseases in any model organism.

GESTALT is based on synthesized DNA barcodes of ∼250 base pairs that contain ten different target sites for CRISPR–Cas9-mediated editing. When a cell containing the barcode is supplied with Cas9 and ten single guide RNAs (sgRNAs) complementary to the target sites, random deletions and insertions accumulate on the barcode with time through non-homologous DNA repair.

Initial experiments in HEK293T cells in vitro showed that a broad range of barcode sequences can be generated, with each mutated barcode corresponding to individual cells. Next, the authors made transgenic single-cell zebrafish embryos carrying the barcode, injected Cas9 and ten sgRNAs into the embryos, and sequenced barcodes from embryos at later developmental stages and from adult tissues. They identified as many as 86–1,323 different barcodes per embryo, and estimated that GESTALT has the potential to generate up to 4,200 unique barcodes.

A maximum parsimony analysis of the barcode sequences revealed that most adult tissues derived from a small number of progenitor cells. Given that groups of similar barcodes were mostly associated with specific organs, the distribution of barcode variations reflected the exclusivity of tissue developmental pathways. These results indicated that information about tissue restriction can be inferred from the barcodes, confirming the validity of GESTALT for recording cellular ancestry.

The large numbers of mutations that accumulate in the barcode greatly expand our ability to learn the history of individual cells in complex organisms. This knowledge can be applied to understanding a range of biological questions, including mechanisms of tissue development, tissue regeneration pathways, disease etiology and the evolution of tumor diversity.

“It's an interesting technique where you actually get the history of the cell, and will have great applications in studying a number of different disease states,” says Leonard Zon, director of the Stem Cell Program at the Children's Hospital Boston, who was not involved in the study. In cancer research, the method should facilitate study of tumor heterogeneity and the development of therapy-resistant cell clones. “Being able to trace that diversity with these lineage tracing techniques is very exciting. It will be interesting to see how this technique applies to metastasis,” says Zon.

Despite the large number of unique barcodes generated by GESTALT, its lineage tracking capacity is not limitless. For instance, the authors note that identical edits can occur in distant lineages by chance, which can lead to false conclusions, and that inter-target deletions can reduce the information-coding capacity of the barcode significantly. Alternative barcode formats could potentially extend the number of possible edits to increase the number of cell divisions recorded.

A logical next step for GESTALT would be to apply the method to adult organisms by engineering conditional expression of Cas9 in adult cells. “Developing a system to induce these genetic changes using CRISPR and then be able to follow the history of adult cells, I think that would be fascinating,” says Zon. “It's just a matter of finding methods to accurately induce Cas9.”