Cancer metastasis is the spread of cancer cells from the original tumor site to a distant site in the body, resulting in a disease that is significantly more complicated and difficult to treat. Despite most localized cancers seeing significant improvements in survival rates over the past few decades, metastasis remains a largely incurable disease, presenting with five year survival rates of well below 20%. Despite accounting for such a massive proportion of cancer related deaths, metastasis remains an extremely poorly understood phenomenon. Tracing the lineage of metastatic cells has the potential to provide a robust means of studying metastasis, allowing for the identification of specific cells that metastasize along with providing the transcriptional identity of those cells.

By using an version of the Genome Editing of Synthetic Target Arrays for Lineage Tracing (GESTALT) method adapted to mammalian systems, known as multiplexed, activatable, clonal and subclonal GESTALT (macsGESTALT), we are able to efficiently track metastatic processes in vivo. This system consists of three components: an inducible Cas9 complex, an array consisting of a series of gRNAs, and a genetic barcode. The barcode is a 250 bp sequence composed of a series of five gRNA target sites each separated by 3 bp protospacer adjacent motif (PAM) sites.

The optimization of gRNA expression is one of the forefront concerns for the application of this system, as the diversity of the barcodes produced is directly dependent on the levels of gRNAs expressed. Premature termination of the RNA polymerase used to transcribe the constructs is predicted to be the primary cause for limited gRNA expression. The 4T sequence in each of the gRNA structures corresponds to a termination efficiency of 75%, and this effect is further exacerbated because the gRNAs are produced in a single transcript, compounding the effect, resulting in the fifth gRNA only being produced 0.098% of the time. 

Introducing an A-T flip into the structure of the gRNA has been found to have no effect on its functionality, and thus serves as a potential means of overcoming premature termination. Cell lines have been produced to test this hypothesis, with gRNAs targeting GFP as a proxy for their expression levels.