CRISPR/Cas9 gene editing has opened the door to numerous biomedical experiments, including studies that systematically deactivate genes in cancer cells to identify the ones that are crucial for the survival and growth of cancer cells. These genes, known as ‘cancer dependencies,’ are often promising targets for drug development. However, recent research indicates that many of these CRISPR screening experiments rely on components called CRISPR/Cas9 guides, which do not function equally well in cells from individuals of various ancestries. This disparity can result in CRISPR screens overlooking important cancer dependencies.
CRISPR/Cas9 gene editing has made possible a multitude of biom rnrnMedical experiments, including studies that systematically deactivate genes in cancer cells to identify the ones that the cancer cells heavily rely on for survival and growth, are a focus of research. These genes, also known as “cancer dependencies,” are often considered as potential targets for new drugs. However, recent findings indicate that many of the CRISPR screening experiments are dependent on components called CRISPR/Cas9 guides, which do not function equally well in cells from individuals of different ancestries. This variability can result in CRISPR screens failing to identify cancer dependencies. These CRISPR guides are brief RNA sequences that direct the CRISPR Cas9 enzyme to a specific location in the genome to cut DNA.The latest research from the Broad Institute of MIT and Harvard reveals that approximately 2% of CRISPR guides fail to target the intended gene, leading to the inability of Cas9 to disable the specific gene. This can lead to a lack of understanding of the potential role of the gene in cancer growth. The study also found that this issue is more prevalent in cells from individuals of African descent, as the CRISPR guides were based on reference genomes primarily from individuals of European ancestry, which does not fully represent the global genetic diversity. These inaccuracies occur in unexpected areas and in ways that were previously unknown.”CRISPR is commonly utilized in preclinical research, but few researchers consider the specific germline and ancestries related to their model systems,” explained Jesse Boehm, an associated scientist at the Broad Institute, and a co-senior author of the paper. “This study demonstrates the importance of conducting a systematic evaluation of the tools and datasets used in order to address hidden biases before they become problematic,” said Rameen Beroukhim, an associate member at the Broad and a co-senior author on the paper, which recently appeared in Nature Communications.The community needs to be aware that functional genomics can be influenced by ancestry bias, which presents an opportunity to further examine this type of data. The team conducted a study analyzing data from the Broad’s Cancer Dependency Map (DepMap), the largest cancer dependency resource. This includes genome-wide screens in over 1,000 cancer cell lines, with about 90 percent from individuals of European or East Asian descent. According to Francisca Vazquez, director of the DepMap at the Broad, less than 1 percent of cell line-guide pairs in the DepMap are affected by the ancestry bias as indicated by this study.dy, it was found that it is important to acknowledge and correct these biases in future libraries. In 2022, the DepMap team removed guide RNAs that were ineffective after the initial results were posted as a preprint. This was done to ensure that the database accurately reflects the lack of sufficient data to draw conclusions, instead of falsely indicating no dependencies for the affected genes.
A new approach to identifying dependencies
Prior to this, the search for cancer dependencies primarily focused on somatic mutations, which are genetic changes that occur in some cells during an individual’s lifetime. However, postdoctoral researcher and lead author of the study, Sean Mis, discovered that In 2020, Misek joined the labs of Boehm and Beroukhim with the goal of investigating how germline genetic variants impact tumor response to treatment. He discovered numerous significant relationships between ancestry and genetic dependencies, with many of these associations stemming from artifacts associated with germline variants. Specifically, Misek observed these effects in CRISPR guides, where the guide RNA sequences did not adequately match the target genetic sequence due to variations depending on ancestry. The scientists determined that 89 percent of these associations were due to germline genetic variants.A recent study revealed that many genome-scale libraries contain mismatches in at least one cell line, particularly in cells from individuals of African descent. Misek, one of the researchers, believes that these biases are likely prevalent in preclinical research and hopes that their findings will spark a larger conversation. Understanding and addressing these biases can be difficult for scientists due to the time-consuming process of gathering necessary data. To combat this, the Broad’s Pattern team created Ancestry Garden, a website utilizing data from various sources.The Genome Aggregation Database (gnomAD) provides a helpful tool for researchers to assess how ancestry may impact their chosen guide. “Many labs utilize CRISPR in some form, and they need a way to verify their reagents,” Misek noted. “Our objective is to make it simpler for individuals to address this problem on their own terms.” Boehm emphasized that genetic variation resulting from ancestry has implications beyond cancer dependency research, and the impact of the team’s discoveries on individual studies will differ. While the bias’s impact was relatively modest.
According to Boehm, while the genetic diversity is limited in the DepMap, it can be significantly larger in experiments focusing on a single or a small number of cell lines.
The study team and DepMap researchers suggest that one way to counter this bias is to enhance the genetic diversity in extensive cell line collections. Vazquez emphasized the importance of addressing this issue and encouraged the community to submit cell lines from underrepresented populations.
