CRISPR progress

Scientists at Cornell University found that Cas3 can delete long sequences of DNA, while a Wellcome Sanger Institute team and collaborators used CRISPR to identify 600 promising gene targets for drug development

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CRISPR/Cas9 is likely the most well-known gene editing combination, but as interest in the field grows, different Cas options are being identified as well, each with their own offerings. One of the latest is Cas3, which, according to a press release from Cornell University's Krishna Ramanujan, “can efficiently erase long stretches of DNA from a targeted site in the human genome … Though robust applications may be well in the future, the new system has the potential to seek out and erase such ectopic viruses as herpes simplex, Epstein-Barr and hepatitis B.”
The work comes out of the lab of Ailong Ke, professor of molecular biology and genetics and a corresponding author of a paper detailing this new CRISPR system, which appeared in Molecular Cell under the title “Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas.” According to Ramanujan's piece, this system offers the ability to sort through the genome for non-coding regions, which comprise 98 percent of the genome and play a role in regulating protein expression in coding genes.
“My lab spent the past ten years figuring out how CRISPR-Cas3 works. I am thrilled that my colleagues and I finally demonstrated its genome editing activity in human cells,” said Ke. “Our tools can be made to target these viruses very specifically and then erase them very efficiently. In theory, it could provide a cure for these viral diseases.”
CRISPR/Cas9 allows researchers to target and cut exact DNA sequences to modify the gene as desired. CRISPR/Cas3, however, can target DNA sequences and then erase DNA for up to 100 kilobases. Ke—along with Yan Zhang, assistant professor of biological chemistry at the University of Michigan and a corresponding author of the paper—and colleagues were able to apply Cas3 to delete DNA sequences of up to 100 kilobases in human embryonic stem cells and in a cell type known as HAP1.
Though the authors note in their paper that gene editing with this type of CRISPR system is programmable, they caution that there is work to do to streamline this approach for the necessary accuracy, with Ke explaining that “We can’t quite define the deletion boundaries precisely, and that is a shortcoming when it comes to therapeutics.”
“With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%–60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site,” the authors reported.
In other recent work, rather than deletion, another team focused on CRISPR research looked at disruption instead. A team of scientists has applied CRISPR to disrupt every gene in 324 cancer models from 30 cancer types to flush out genes that play a role in cancer survival. The Wellcome Sanger Institute team was joined by collaborators from GlaxoSmithKline, Open Targets, EMBL-EBI and other organizations.
They worked to disrupt nearly 20,000 genes, making it one of the largest CRISPR screens of cancer genes to date, according to a Wellcome Sanger Institute press release. The cancer types screened included breast, lung, colon, ovarian and pancreatic, among others, and after identifying several thousand key genes, the team narrowed their findings down to roughly 600 genes that had the most potential as drug targets. The team's paper, “Prioritization of cancer therapeutic targets using CRISPR–Cas9 screens,” appeared in Nature.
Among the genes that made the “short list” was Werner syndrome RecQ helicase (WRN), which appeared in several of the screened cancer types. Cancer cells that presented with a damaged DNA repair pathway need WRN to survive. These cancers are also known as microsatellite unstable cancers, and microsatellite instability is seen several cancers, including 15 percent and 28 percent of colon and stomach cancers respectively.
“What makes this research so powerful is the scale. CRISPR provides a unique tool to accelerate discovery of oncology drug targets, and this study is a salient leap in a positive direction,” said Prof. Karen Vousden, chief scientist at Cancer Research UK. “But we should remember that studying cells in the lab doesn’t always reflect the complexities of cancer in the human body and so will not necessarily reflect how someone will respond to a drug. This work provides some excellent starting points and the next steps will be a thorough analysis of the genes that have been identified as weaknesses in this study, to determine if they will one day lead to the development of new treatments for patients.”
This effort is one of many being undertaken by the Wellcome Sanger Institute related to the Cancer Dependency Map, a strategic collaboration with the Broad Institute of MIT and Harvard that is combining “the work of multiple experimental and computational research project at the Sanger Institute with the shared aim of identifying dependencies in cancer cells which could be exploited to develop new therapies. This knowledge is foundational for our understanding of cancer biology and the development of precision cancer medicine.”
“The Cancer Dependency Map is a huge effort to identify all the weaknesses that exist in different cancers so we can use this information to empower the next generation of precision cancer treatments. Ultimately, we hope this impacts on the way we treat patients, so many more patients get effective therapies. In the meantime, this tool will be freely available for scientists across the world to understand what makes a cancer a cancer, and how we might target different types of cancers much more effectively than we do today,” remarked Dr. Mathew Garnett, co-lead author from the Wellcome Sanger Institute and Open Targets.

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