Lauren Gatta

In a new study, researchers at Yale have created a new method to detect metastasis, or the spread of cancer, by identifying the genetic interactions between cells.

Metastasis, a complex process regulated by multiple genes and potentially involving more than 100,000 interactions between genes, is a leading cause of death among patients with solid tumors. Using CRISPR gene editing technology and parallel computing, this study identifies genetic targets that have been implicated in metastasis and introduces a useful tool that can be applied in other areas of cancer research.

“[This technique] doesn’t only have to be used to study metastasis. We can try different cells and different organs. It can also be used to study genetic dependencies in a more general use,” said Ryan Chow MED PHD ’22, first author of the study.

Senior author Sidi Chen, professor of genetics at the School of Medicine, and his team of researchers created a genetic “library,” or a collection of DNA, of more than 12,000 unique combinations. These combinations of DNA can silence gene expression in a cell and stop the production of the protein that the silenced gene encodes for — a process known as a “knock-out.” The study observed the effect of “knocking out” different combinations of two genes, or a double knock-out.

“The 12,000 combinations refer to unique CRISPR constructs that targeted 385 candidate genes. We can determine how a specific gene is targeted by changing the RNA sequence, which is the guide where the CRISPR enzyme will cut,” Chow said. “The RNA sequences guiding the enzyme to the proper location are picked by looking at the genome, and we design the RNA so that it is complementary to the DNA sequence of the particular location in the genome.”

CRISPR refers to a specialized segment of DNA originally found in bacteria, which are used in tandem with “molecular scissor proteins” like Cpf1 or Cas9, to defend against viral infections by cutting the DNA of viral invaders. These “scissor” proteins can cut DNA in other organisms, recombining different segments of DNA — a process referred to as gene editing.

Although the Cas9 protein is better known than Cpf1, Chow said that the choice of Cpf1 enabled multiple gene knockouts, which was key to studying the interaction of multiple genes in this study.

“Unlike Cas9, Cpf1 has the unique property of doing multiple knockouts with a single RNA array, which makes it easier to do multiple knockouts that we used for analysis in our study,” he said. “Historically, it’s been pretty challenging to do this — it’s pretty hard to do targeted mutations in multiple genes at the same time. But now we’ve demonstrated that we can use Cpf1 to see interactions between multiple genes.”

The new analysis method — which the researchers termed “massively parallel CRISPR-Cpf1/Cas12a crRNA array profiling,” or MCAP — allows for many different combinations of double-knockouts to be statistically evaluated at the same time. These parallel, automated computations that happen simultaneously increase the efficiency of analysis, which is a goal of high-throughput methods in biology.

The study found that several pairings of mutant gene knockouts caused aggressive metastasis in mice. In addition, Chow noted that many of the genes involved in metastasis and cancer are related. Nonetheless, some of the identified gene pairs were found to specifically promote the spread, and not formation, of cancer.

CRISPR was first discovered in 1993.

Viola Lee | kyounga.lee@yale.edu