These days, it doesn’t take a rocket — or genome — scientist to recognize that our capacity for exploring genomes is at an all-time high. In laboratories here at The Jackson Laboratory (JAX) and across the world, ever-powerful machines churn out endless strings of genetic information; increasingly precise tools home in on specific spots in the genetic code and can even make corrections; and robust analytical methods help make sense of a morass of data. Propelled by these capabilities, scientists are uncovering clues within our DNA that shed light on the mysteries of biology and help unravel the complexities of disease.
Behind the scenes, technologies that fuel this mind-bending pace of discovery are buzzing and humming away. Three methods in particular are worthy of notice: high-throughput genome sequencing, CRISPR, and single-cell genomics. Here, we offer a brief primer on these approaches, describing how they emerged, how they work, and how they are transforming biology and medicine.
The tools for reading — or “sequencing” — the chemical letters that make up our DNA have evolved rapidly over the last decade. Researchers can now gather information more quickly and at a lower cost than ever before. In fact, sequencing costs have been on a veritable free fall for the last several years, even outpacing a well-known trend in the computer hardware industry, called Moore’s Law, in which computing power increases (and costs drop) two-fold every two years. (Check out these graphs, courtesy of the National Human Genome Research Institute, which plot the decline in DNA sequencing costs relative to Moore’s Law.) Although Moore’s Law wasn’t conceived with biotech in mind, it has become a common benchmark for measuring technology performance and growth.
What’s behind this rapid change? Well, in short, entirely new ways of reading DNA that diverge from the standard “first-generation” approach, known as the Sanger method. Named for its inventor, Frederick Sanger, this kind of sequencing was the scientific workhorse of the Human Genome Project (HGP), a sweeping, international effort to decode the full human genetic blueprint, which culminated with the publication of an initial draft genome sequence in 2001.
In order to determine the order of chemical letters (or “bases,” abbreviated as A, C, G, and T) that make up the genome, the Sanger method creates new copies of the DNA target of interest. The raw material for these copies comes from bases that carry special modifications, such as fluorescent tags that glow a different color depending on the type of base — for example, green for A, red for T, and so on. As these modified bases are incorporated into the newly synthesized DNA strand, it becomes possible to decipher the sequence. (Watch this short video, which explains the basic idea behind Sanger sequencing.)
Fueled in part by the HGP, scientists continually tweaked the Sanger method, improving its performance, automating it, and generating ever-longer strings of DNA sequence (called “reads”). While this enhanced the genomic bang-for-the-buck, eventually the technology hit a wall: The typical Sanger sequencing reads tend to top out at 800 to 1000 bases. With a length of roughly 3 billion bases, the first full human genome sequence took about ten years and nearly $3 billion to complete.
If sequencing whole genomes was to become more commonplace (and take less time and money), an entirely new approach was needed.
Enter so-called “next-generation” sequencing (NGS), which relies on different kinds of chemistry than Sanger sequencing. While there are multiple NGS methods that each differ in the nitty-gritty details, they all share a handful of key properties. First, instead of aiming to maximize read length, these methods all yield fairly short bits of DNA sequence, from as short as 50 bases to a few hundred bases. Second, next-gen technologies essentially miniaturize the sequencing process — decoding a piece of DNA within a very, very tiny space, allowing many other pieces of DNA to be sequenced simultaneously. (This is why NGS is often called massively parallel sequencing.)
There are even third-generation technologies that take yet another approach — passing a single molecule of DNA through a tiny opening (a so-called “nanopore”) and determining the identity of each base as it passes through the pore by virtue of a change in electrical activity.
The combined effect of these new technologies is that sequencing costs have fallen dramatically. And driving down costs means scientists get more DNA sequence for their laboratory dollar. Now, it is economically feasible to sequence not one genome, but hundreds of them, even many thousands.
This evolution has ushered in a new era of biomedicine, in which it is possible to probe the human genome on an individual level, revealing variations in one person’s DNA that may have significance for understanding disease biology and could even guide treatment. Countless laboratories here at JAX are harnessing these new capabilities. For example, Charles Lee, professor and scientific director at The Jackson Laboratory for Genomic Medicine, is a world leader in applying sequencing and other genome-scale technologies to reveal how individual genomes vary from one another. He is part of an international consortium that just reported the completion of a major project, the 1000 Genomes Project, which sequenced the genomes of over 2,000 people from 26 populations across the globe. Lee also led a recent study that used DNA sequencing to explore the tumors of more than 100 people with gastric cancer, unveiling mutations in genes that could prove to be key drug targets in this form of cancer. These discoveries, and many others, underscore the power of second- and third-generation DNA sequencing technologies to push the frontiers of knowledge in biology and medicine.
If sequencing is like reading DNA, then CRISPR provides the power to edit — to correct the typos, or “mutations,” that can arise in genomes — and to do so with an unprecedented level of precision. This genome-editing technology has taken the scientific world by storm with its breakneck pace of growth: The approach was first shown to work in mouse and human cells less than three years ago and has already been applied to a range of biological systems and disease areas. Indeed, it has captivated researchers’ imaginations with the remarkable opportunities it opens up in the laboratory and soon, perhaps, the clinic.
CRISPR’s power stems not only from its precision, but also its ease of use. Genome-editing experiments that previously took months, even years, to complete can now be done in a fraction of the time. Combined with the recent growth of DNA sequencing, which has led to a dramatic rise in the number of genes and gene mutations associated with disease, CRISPR packs a powerful, one-two punch — giving scientists the tools to study the biology behind these mutated genes and to correct them.
Short for Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR’s name reflects its beginnings: a collection of DNA sequences, unusual for their highly repetitive nature. These sequences were first found in the bacteria Escherichia coli in the late 1980’s and garnered little fanfare. But as more and more microbes’ genomes were sequenced, these strange repeats kept popping up. First, they were dismissed as genomic junk, but researchers later came to appreciate their importance: tiny snippets of DNA left behind by pathogens (specifically viruses) that had infected the bacteria. These viral remnants are a physical record, forming a kind of primitive immune system that enables bacteria to defend themselves against future attacks.
In the course of probing the ins and outs of this type of bacterial immunity, two important features emerged. First, one of the CRISPR systems includes an enzyme, called Cas9, which can cut DNA. Second, the system is programmable, meaning it can be directed to precise spots in the genome by virtue of a special guide molecule made of RNA. These discoveries helped launch what has become arguably the hottest technology in biomedicine since the dawn of recombinant DNA.
Although CRISPR is indeed remarkable, the technology is new and underexplored. While there is intense interest — and concern — about its therapeutic use in humans, it is a revolutionary tool for research. JAX assistant professor Haoyi Wang is a leader in the development and use of genome editing tools, including CRISPR, as well as other methods. He helped develop CRISPR/Cas9 while working as a postdoctoral associate in Rudolph Jaenisch’s lab at MIT. One of his areas of focus here at JAX is to apply it in mice — including ways to streamline the development of genetically modified strains, making the process not only quicker but also less expensive. So, while the scientific community plots a careful and measured course with respect to CRISPR’s applications in the clinic, the genome-editing tool will surely continue to blaze new trails in the laboratory.
The human body is made up of nearly 40 trillion cells and roughly 200 different cell types. Amidst this significant diversity, scientists have typically explored cells in bulk. Rather than examining just a single cell, researchers analyze thousands or millions at a time. And that means what can be gleaned from an experiment usually reflects an entire population of cells, rather than one particular cell.
One reason for this lack of individuality is technical — the amount of DNA (or RNA or protein) that can be extracted from a single cell is often not enough to support genome-scale analyses. Yet there are big questions in biology that stem from single cells. Cancer, for example, begins when the DNA of a cell is damaged (or “mutated”) in such a way that allows it to grow out of control, leading to many other rogue cells and the formation of tumors. But cancer isn’t the only area where a deep knowledge of cells as individuals could be beneficial. The brain, the immune system, blood — simply put, many, if not all, of the body’s systems are built on the concept of cellular diversity. And understanding how this diversity is programmed, through changes in DNA, RNA and beyond, is an essential piece in the vast puzzle of human biology.
Recent advances in the techniques for isolating single cells, together with methods for amplifying their genetic material, now make it possible to explore the genomes of single cells. With the birth of this new field, aptly known as single-cell genomics, scientists can probe the full complement of DNA (or even RNA) that exists within a cell.
Recognizing the remarkable opportunities to apply single cell technologies to major questions in biology and medicine, JAX recently launched a joint center for single cell genomics together with the University of Connecticut, including UConn Health. Paul Robson, who serves as JAX Genomic Medicine's director of single cell genomics, believes the work of the new center will be critical to advancing the goals of precision medicine: “If you want better insight into how biology works, you need to look at its fundamental unit,” he says.
Although the field of single cell genomics is still fairly new, it is already becoming clear that cells once thought to be genetically similar, if not identical, are in fact quite different. For example, cancer cells are not the only cells that can acquire changes in their DNA. These kinds of somatic (that is, non-inherited) mutations also appear in neurons, and could play a role in epilepsy, autism and other disorders of the developing brain. It is also possible that these genetic differences are somehow required for normal neuronal development. To be sure, we have only just scratched the surface in identifying and understanding the diversity that lies within our cells.
Indeed, as we ride the waves of single cell genomics — and genome sequencing and editing, too — we can see further than ever before. That means our biomedical knowledge is more advanced and more precise than ever, and it’s growing at a phenomenal pace. At the same time, we must also remember that there is still much more to learn.
Nicole Davis, Ph.D., is a freelance writer and communications consultant specializing in biomedicine and biotechnology. She has worked as a science communications professional for nearly a decade and earned her Ph.D. studying genetics at Harvard University.