New CRISPR-Based Map Opens Door to New Genetic Testing, Therapeutic Possibilities

Researchers from all around the world have been working on mapping human genes to their functions. Now, for the first time, that mapping has been completed, and it opens the doors for all kinds of genetic testing and medicinal possibilities.

In 2003, members of the Human Genome Project completely sequenced the human genome, but the work wasn’t done. Ever since the human genome was sequenced, Massachusetts Institute of Technology professor Jonathan Weissman, Ph.D. and his colleagues have been creating a comprehensive functional map targeting all expressed genes with CRISPR interference (CRISPRi) across more than 2.5 million human cells. Now, the researchers have published their genome and phenome research in Cell.

One of the major goals of the project was to define the relationship between genotypes (the genes in DNA responsible for a characteristic) and phenotypes (the non-inherited, visible expression of that gene). A map of those relationships could drastically change the way researchers conduct genetic experiments.

“Rather than defining ahead of time what biology you're going to be looking at, you have this map of the genotype-phenotype relationships and you can go in and screen the database without having to do any experiments,” Weissman told MIT News.

Weissman and his colleagues tackled this question using the Perturb-seq approach, a method using CRISPR-based screens with single-cell RNA-sequencing readouts. The method uses CRISPR-Cas9 genome editing to make genetic changes to cells, then uses RNA sequencing to see how the RNA is expressing that genetic change, making it possible to determine the impact of turning genes on or off. Using this approach, researchers may be able to better understand a range of biological underpinnings to many cancers and other high-need conditions.

“Forward genetic screens are powerful tools for the identification of cancer dependencies, essential cellular machinery, differentiation factors and suppressors of genetic diseases…. Once a phenotype is defined, the genotype-phenotype map can be used to explore its genetic underpinnings, in a manner analogous to a forward genetic screen, as well as its relationship to other cellular phenotypes,” Weissman et al wrote in the Cell paper. 

Weissman and his associates, including MIT professor Aviv Regev, Ph.D., published a paper on the Perturb-seq method in 2016, but it was limited in the sets of genes it could be used on, and it was prohibitively expensive.

However, researchers continued searching for a way to make the process scalable, including pioneers such as Joseph Replogle, an M.D.-Ph.D. student in Weissman’s lab and later co-founder of the publication in Cell; Britt Adamson, Ph.D. an assistant professor in the Department of Molecular Biology at Princeton University; former Weissman Lab postdoc Tom Norman, a co-senior author of the paper who now leads a lab at Memorial Sloan Kettering Cancer Center; and a group at 10x Genomics. Together, they created a model for making Perturb-seq experiments more scalable. They published a proof-of-concept paper in Nature Biotechnology in 2020.

Eventually, Replogle, graduate student Reuben Saunders and other researchers in Weissman’s lab found a way to scale up the process to the entire human genome. They performed Perturb-seq across more than 2.5 million cells.

As the researchers collected the data that was published in Cell, they made several interesting discoveries. One was that aneuploidy - a condition of having an abnormal number of chromosomes - was influenced by a specific subset of genes. Aneuploidy has previously not been studied often because it requires a single-cell readout, which is difficult but now possible with the Perturb-seq method.

Another insight from the study is that mitochondrial genome expression has stress-specific regulation.

“Although previous studies have described distinct regulation of the mitochondrial genome in response to specific perturbations, our data generalize this phenomenon to a comprehensive set of stressors,” Weissman et all said.

Weissman and his colleagues are making it available on an MIT webpage so others can build on their research.

“This really is the culmination of many years of work by the authors and other collaborators, and I’m really pleased to see it continue to succeed and expand,” Norman told MIT News.