Some CRISPR screens may miss cancer drug targets

CRISPR/Cas9 gene editing has made possible a host of biomedical experiments, including studies that systematically turn off cancer cell genes to find those that cancer cells heavily rely on to survive and grow. These genes, or “cancer addictions,” are often promising therapeutic targets. But new research shows that many of these CRISPR screening experiments rely on components, called CRISPR/Cas9 guides, that don't work as well in cells from people of all backgrounds, which can cause CRISPR screens to miss the cancer addictions.

These CRISPR guides are short RNA sequences that direct the CRISPR enzyme Cas9 to a specific site in the genome to cut DNA and turn off a targeted gene. The new findings, by scientists at the Broad Institute of MIT and Harvard, show that about 2 percent of these guides miss their target. This means that Cas9 will not cut and deactivate a specific gene, thereby masking that gene's potential role in cancer growth. The team found that this occurs disproportionately in cells from people of African ancestry, because the CRISPR guides were designed using reference genomes from people largely of European ancestry and do not represent not fully global genetic diversity.

“These inaccuracies exist in places we may not recognize and in ways we would not have predicted,” said Rameen Beroukhim, an associate fellow at the Broad and co-senior author of the paper, which appeared recently in Natural communications. “This work shows that it is definitely worth carrying out a systematic evaluation of all the tools and datasets we use so that we can correct these hidden biases before they become a problem.”

“CRISPR is used everywhere in preclinical research, but only a minority of researchers think carefully about the specific germline and ancestry related to their model systems,” added Jesse Boehm, associate scientist at the Broad and co-senior author of the study. study. paper. “This is a warning to the community that functional genomics is not immune to ancestry bias, and a source of opportunity to take a closer look at this type of data.”

In their study, the team analyzed data from Broad's Cancer Dependency Map (DepMap), the largest cancer dependency resource, which currently includes genome-wide screens of more than 1,000 cancer lineages. cancer cells, about 90% of which come from people in Europe or Europe. East Asian descent.

Francisca Vazquez, director of DepMap at the Broad, said less than 1 percent of DepMap guide cell line pairs are affected by the ancestry bias shown by this study, but it is important to recognize and correct these biases. in future libraries. . After these results were first published as a preprint in 2022, the DepMap team removed all non-working guide RNAs from their library, so that instead of falsely returning no dependencies for genes assigned, the database indicates that there is not enough data. to draw conclusions.

A new type of addiction research

Previously, cancer addiction research focused on genetic changes that occur in certain cells over a person's life, called somatic mutations. But when postdoctoral researcher and first author of the study, Sean Misek, joined Boehm and Beroukhim's labs in 2020, he wanted to know how germline genetic variants — which are inherited and present in every cell in the body — influence how tumors respond to treatment.

Misek discovered many strong associations between ancestry and genetic dependencies, and that most of these associations came from artifacts related to germline variants. He notably observed these effects in the CRISPR guides. The sequence of the guide RNAs did not sufficiently match the target genetic sequence because this target sequence varied depending on ancestry.

Scientists found that 89 percent of guides in genome-wide libraries have a mismatch in at least one cell line. They also found that mismatches occur to a greater extent in cells from people of African descent.

“This kind of experimental bias is probably pervasive in preclinical research,” Misek said. “We hope this paper is part of a broader conversation.”

Understanding the extent of this bias in a research project can be difficult for a scientist, as it can take several days to download all the necessary data. To address this problem, Boehm, Beroukhim and the Pattern team at the Broad created Ancestry Garden, a website based on data from the Genome Aggregation Database (gnomAD) that can help researchers determine the effect of ancestry on a guide of their choice.

“Many labs are using CRISPR in one way or another, and they should have a mechanism to verify their reagents,” Misek said. “Our goal is to make it a little easier for people to alleviate this problem on their own.”

Library lessons

Boehm said genetic variation due to ancestry affects research far beyond cancer addiction research, and the extent to which the team's findings will impact individual studies will vary. Although the effect of this bias was relatively modest in the DepMap, it can be much larger in experiments that study only one or a small number of cell lines, Boehm said.

Moving forward, the study team and DepMap researchers say an important way to address this bias is to increase genetic diversity in large-scale cell line libraries.

“We encourage the community to send us cell lines from underrepresented populations if they have them,” Vazquez said. “This is a very important question to resolve.”