Genes are like Egyptian hieroglyphs. Thanks to advances in whole genome sequencing, it’s getting easier to read every letter of DNA. But the strings of A, T, C and G bring up a second puzzle: what do they mean?
It’s a problem that has haunted biologists since the completion of the Human Genome Project. By using our basic genetic code, the project hypothesized, we would be able to master control of hereditary diseases, manipulate them at will, and easily predict the consequences of each gene that laid the foundation. for our bodies, our functions and our lives.
The vision didn’t quite work out. DNA sequences, while they capture extremely powerful genetic information, don’t necessarily translate to indicate how our bodies behave. Genes can switch on or off in different tissues, depending on the need of the cell. Reading a DNA sequence for any gene is like parsing the basic code of a cell’s internal program. There is the raw genetic code – the genotype – that determines the phenotype, the software of life that determines how cells behave. Linking the two has taken decades of painstaking experimentation, slowly building an encyclopedia of knowledge decoding a gene’s influence on biological functions.
A new study stepped up the effort. Led by Drs. Thomas Norman and Jonathan Weissman of Memorial Sloan Kettering Cancer Center in New York and the University of California, San Francisco, respectively, the team built a Rosetta Stone for translating genotypes into phenotypes, using CRISPR.
They went big. By altering gene expression in more than 2.5 million human cells, the technology, called Perturb-seq, extensively mapped how each genetic disruption changes the cell. The technology revolves around a kind of CRISPR on steroids. Once introduced into cells, Perturb-seq quickly alters thousands of genes — a brutal shake-up at the genomic scale to see how individual cells respond.
In other words, Perturb-seq is a large-scale tool that can help scientists translate DNA code into function — a Rosetta stone to uncover the inner workings of our cells. Years in the making, the dataset is open to anyone to explore.
“I think this dataset is going to enable all kinds of analysis that we haven’t even figured out yet by people coming from other parts of biology, and suddenly they just have this available to draw from,” Norman said.
Lost in translation
What is the function of a gene? It’s easy to think that genes are your destiny, but that’s far from the truth. Environmental factors, such as a huge bowl of spaghetti or a walk along the beach, can easily alter gene expression, bodily functions, and possibly your mind and body.
If so, what’s the point of sequencing whole genomes if the outcome is always in motion? “A central goal of genetics is to define the relationships between genotype and phenotype,” the authors said. In other words, what does a gene mean? actually do?
Scientists have long sought a bridge between genotype and phenotype. It is a laborious process. For example, one method disrupts genes that may be related to a condition one at a time and observes the behavior of the cells. The idea, called “forward genetics,” is gene targeting rather than focusing on the phenotype. An alternative approach, “reverse genetics,” delves deep into how a body or mind changes with a specific genetic edit.
Each method is an uphill battle. With more than 20,000 genes in our bodies and each cell behaving slightly differently (even with the same genetic changes), deciphering a gene’s function often takes years, if not decades.
Is there a way to speed up the process?
The CRISPR Rosetta Stone
Enter CRISPR. Long revered as a genetic editing multi-tool, the method has further evolved into a biological translator. At its core is a technology called Perturb-seq, first published in 2016 to parse the expression of genes. Perturb-seq makes it possible to monitor the consequences of turning a gene on or off in a single cell. The method quickly became famous in 2020 for its efficiency at changing multiple genes at once.
It’s a huge win for cell biology, the team said. While scientists have easily broken down the huge web that connects genes and proteins, it has been a struggle to pin down the role of individual genes. “We often take all the cells where ‘gene X’ is turned off and put them together to see how they’ve changed,” Weissman says. “But sometimes different cells that lose that same gene behave differently when you knock down a gene, and that behavior can be missed on average.”
The idea behind Perturb-seq is quite simple. Imagine a toddler breaking things and realizing what he did after seeing the consequences. Perturb-seq uses CRISPR-Cas9 to silence multiple genes at once, which can sometimes change a cell’s behavior. While it was a powerful tool, it was difficult to scale, studying at most a few hundred genetic perturbations at a time for predefined biological questions.
So why not extend the method to the whole genome?
“The advantage of Perturb-seq is that you can get a large dataset in an unbiased way,” Norman says. “Nobody knows exactly what the limits are of what you can get from such a dataset. Now the question is: what do you actually do with it?”
The life of a cell
In the new study, the team found the magic sauce for making genome-wide changes in human cells with CRISPR for the first time. An important point was the optimization of a library of guide RNAs (sgRNAs), the ‘bloodhounds’ that track a gene. They then captured cells infected with CRISPR and analyzed their gene expression. Overall, the team focused on nearly 2,000 genes. By cross-referencing altered genes with each cell’s phenotype, they clustered genes into networks linked to a cellular outcome.
One puzzling gene stood out: C7orf26† Combining it with CRISPR changed the way a cell builds a huge molecular complex called the Integrator, which helps make molecules that control gene activity. Before Perturb-seq, C7orf26 had never been associated with the complex before.
In another analysis, the team found a subset of genes that alter how “daughter cells” inherit the parent genome. For example, removing some genes changed the distribution of chromosomes as a cell divides. Adding or removing a chromosome can fundamentally change our biology, for example by leading to Down syndrome.
For Norman, this aspect is the most interesting part of Perturb-seq. “It captures a phenotype that you can only get with a single cell readout. You can’t help but go after it.”
This database is just the beginning. The team wants to use Perturb-seq on other human cell types and all data is available for collaboration. With the emergence of Ultima Genomics, an ultra-low-cost genomic sequencing solution, single-cell CRISPR displays are likely to play an even greater role in biotechnology, such as in analyzing the genomes of iPSCs (induced pluripotent stem cells).
In fact, for Weissman, it may cause a shift in the way we approach cellular mysteries. “Instead of pre-defining what biology you’re going to look at, you have this map of the genotype-phenotype relationships, and you can go into the database and screen without doing any experiments,” he said.
Image Credit: Jen Cook/Chrysos Whitehead Institute