Genetic Engineering & Biotechnology News

DEC 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Page 55 of 69

22 | DECEMBER 2017 | CRISPR technology." "The Essner, McGrail, and Dobbs laboratories, in collaboration with the Clark and Ekker laboratories, created specialized vector technology that enables precise knockins. The key is in the design of the short homology arms. Because the arms are 24 to 48 bases long, they ensure high integra- tion efficiency. Altering donor homol- ogy lengths by as little as one base pair can double knockin efficiencies." The technology is called pPRISM, for PRecise Integration with Second- ary Markers. It contains sites for cloning gene homology arms, options for several colors of fluorescent tags, or a stop codon. Homology arms are designed to match the sequences directly upstream and downstream of a CRISPR genomic DNA target site (Figure 2). The Iowa State University/Mayo Clinic team also designed a "universal gRNA" that can efficiently liberate the knockin cassette from the vector and expose the regions of homology. pPRISM is optimized for the Golden Gate cloning method, where multiple DNA fragments can be directionally assembled into a vector backbone in a "one-pot" reaction. In vivo targeting with pPRISM and CRISPR/Cas9 di- rects precise knockin with little mosa- icism in up to 80% of injected animals. Welker uses the technology to study connexin 43.4, a membrane protein involved in intercellular com- munication and left/right develop- ment. "The pPRISM technology enabled knockin of specific connexin 43.4 alleles tagged with different fluo- rescent markers," says Welker. "We can then follow each allele to ascertain its effect on heart laterality." The Essner and McGrail laborato- ries successfully targeted six loci in the zebrafish genome and will make this technology available to the communi- ty. "We expect our vectors, protocols, and web interface for homology de- sign to streamline experimental design and broaden the use of CRISPR for homology-based gene editing," adds Welker. Natural Killer Cell Therapy in Action Natural killer (NK) cells, cytotoxic lymphocytes of the innate immune system, identify target cells—virally infected cells and cancer cells—by recognizing complex patterns of mem- brane receptors. Once they have iden- tified their targets, the NK cells live up to their name. Multiple therapeutic approaches have attempted to harness NK cells. For example, clinical trials have been initiated to determine whether adop- tive cellular transfer of allogeneic NK cells could be effective against solid tumors such as neuroblastoma, renal, colon, gastric, and ovarian cancers. In these trials, NK cells tend to disap- point. They may lack efficacy because they are negatively downregulated by the tumor microenvironment. "If we are able to knockout these negative regulators, we can create an NK cell that is equipped to fight can- cer," says Emily Pomeroy, a graduate student in the laboratory of Branden Moriarity, Ph.D., a pediatric oncolo- gist at the University of Minnesota. "Combined with easy expansion and well-developed adoptive transfer, such modified NK cells could become a single source of immunotherapy to treat a broad range of cancer types." The research team, led by Dr. Mo- riarity, used targeted genome editing to knockout genes in primary immune cells, including NKs, which are notori- ously difficult to edit. (As part of this effort, Pomeroy helped to develop a transfection protocol. Specifically, she led work to define electroporation conditions that are capable of pre- serving cell viability while maintain- ing 95% transfection efficiency.) Five known downregulator genes were selected, and knockouts were fully characterized. The team plans to put together a combination of edited NK cells to achieve maximum antitumor efficien- cy. "A significant advantage of NK cell therapy is that no matter how potent it is, the transplanted cells do not per- sist," added Pomeroy. In comparison with antibody blockade of inhibitory receptors, another considerable advan- tage of CRISPR-mediated gene editing lies in its ability to knockout intracel- lular proteins. Dr. Moriarity's team has already created multiplex knockouts. Going forward, it hopes to expand on its work with CRISPR-edited NK cells. Soon, these cells may be evaluated in vivo, in studies using human cancer cell lines xenographed into mice. Ultimately, it is feasible that the team's work could find its way to the clinic. B E S T O F C R I S P R 2 017 Using CRISPR to Improve Disease Modelin g Continued from page 21 While there are numerous approaches to implement CRISPR/Cas9 gene editing, many of them experience a relatively high rate of off-target effects, limiting the system's applicability.

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