It was the curiosity of two basic scientists, structural biologists Jennifer Doudna, who is now a quasi-celebrity, and Jillian Banfield, an environmental researcher at Berkeley that led to one of the most impactful discoveries of modern biology: the use of CRISPR-Cas9 as tool to edit the genetic code. The story begins when they set out to study the genetic code of an obscure bacteria species that lived in a highly acidic abandoned mine. They found the genomes of these microbes to contain unusual repeating sequences called “clustered regularly interspaced short palindromic repeats,” or CRISPR. Doudna would later learn that these sequences were part of a bacterial adaptive immune system that fends off viruses, after a fortuitous conversation with Emmanuelle Charpentier, a French microbiologist, at a conference. Dr. Charpentier had already been studying the role of CRISPR as an adaptive immune system in bacteria. But what if this cellular defense system can be manipulated to edit genomes? It was an eureka moment. The rest is history.
Since the initial discovery in the early 2000s, many advances have been made in our understanding of how CRISPR-Cas9 works and how it can be manipulated for genome editing. CRISPR-Cas9 has since developed into one of the most powerful tools in molecular biology. Fundamental to the function of CRISPR-Cas9 is the reliance on small guide RNA sequences for the specific detection of target DNA. Researchers at the Bradley lab (Basic Sciences Division) at Fred Hutch wondered if CRISPR-Cas9 could be the answer to another very fundamental question in biology, one that other gene editing techniques fell short of answering: what is the functional significance of poison exons that disrupt their host genes’ reading frames yet are frequently ultraconserved? In a recently published paper in Nature Genetics, researchers from the Bradley lab described a new method which they affectionately called pgFARM (pronounced pig farm) which is short for “paired guide RNAs for alternative exon removal”.
Dr James Thomas, a postdoc in the Bradley lab, led the study. Dr. Thomas explained the significance of this new technology: “pgFARM is the first method for multiplexed functional screens of alternative splicing. Instead of knocking out entire genes, we can do functional genomics with isoform level resolution. It also enabled us to identify previously unknown roles for ultraconserved elements in the human genome. We specifically targeted a class of these highly conserved elements called “poison exons” and found many of them to be essential for cell growth in vitro and in vivo.” The researchers sought to test the role of poison exons in tumorigenesis. They collaborated with Dr. Alice Berger (Human Biology Division) to test the effect of poison exons in lung tumor cells. Dr. Thomas elaborated on the findings of the study, “we found that several poison exons have tumor suppressor activity in vivo. In other words, knocking out these poison exons causes tumors to grow faster in mice.”
To study ultraconserved alternative exons, Dr. Thomas and colleagues needed a tool to experimentally manipulate them. Using a pair of guide RNAs for the CRISPR-Cas9 system instead of one was the key to the development of this technique. Dr. Thomas recounted the story that inspired the project: “We were originally inspired by a paper from Jay Shendure’s lab (University of Washington). A student named Molly Gasperini used paired gRNAs to delete regulatory elements. We thought we could adapt that technique to delete specific exons to manipulate splicing. The project really began after I met with her and she taught me how to use the system.” It was yet again one of those fortuitous encounters in science!
In the future, the Bradley lab will explore the role of poison exons in early embryogenesis. Dr. Thomas explains, “Previous studies generated ultraconserved element knockout mice and found no viability or postnatal defects. That was really surprising!” The Bradley lab is already working on making poison exon knockout mice. “We have data suggesting that some ultraconserved poison exons are important for viability,” added Dr. Thomas.
This work was supported by funding from the Washington Research Foundation, the Leukemia and Lymphoma Society, Edward P. Evans Foundation and the National Institutes of Health.
UW/Fred Hutch Cancer Consortium members Robert Bradley and Alice Berger contributed to this work.
Thomas JD, Polaski JT, Feng Q, De Neef EJ, Hoppe ER, McSharry MV, Pangallo J, Gabel AM, Belleville AE, Watson J, Nkinsi NT, Berger AH, Bradley RK. 2020. RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons. Nature Genetics.