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 58 of 69 | DECEMBER 2017 | 25 ➜ Medical School, work was undertaken in his laboratory on PK15 porcine kidney epithelial cells to determine if PERVs could be eradicated. It was crucial to avoid disrupting the envelope gene and the terminal regula- tory elements, as both of these could be important during normal pig fetal growth. In addition, a highly con- served target in the viral polymerase gene was desired for the guide RNA (gRNA) to bind. First, the copy number of PK15 PERV was determined to be 62. Then, when CRISPR/Cas9 was used along with two gRNAs, one which did the bulk of the work, all 62 copies of the PERV pol gene were disrupted, dem- onstrating the possibility that PERVs could be inactivated for potential clini- cal pig-to-human xenotransplantation. The repeats were well separated, and not clustered, which could have meant higher toxicity. After two weeks of cell culture, about 8% of clones were 100% al- tered, and no rearrangements were found. Although a few off-target ef- fects and point mutations were expect- ed, they were deemed unlikely to have an impact on pig fetal development. As with conventional breeding, PERV- free clones were empirically selected as they were the healthiest. In addition to disrupting dozens of endogenous viral elements, Dr. Church's group altered dozens of genes involved in immune and blood- clotting functions to increase human compatibility. Some of the changes were so extensive that more powerful DNA recombination tools, and not CRISPR, were utilized. This work may benefit eGenesis, a Cambridge biotech focused on lever- aging CRISPR technology to deliver safe and effective human transplant- able cells, tissues, and organs. eGen- esis was cofounded by Dr. Church and Luhan Yang, Ph.D., in early 2015 and is based on their research. Ex Vivo Indications Another emerging company B E S T O F C R I S P R 2 017 Demand for obtaining genetically engineered animal models on faster timelines has fueled the development of gene-editing technologies like CRISPR. As interest skyrockets, it is critical to evaluate how and where to employ CRISPR in the development of animal models for in vivo studies. The simplicity and efficiency of CRISPR, and the promise of shorter lead times, have led investigators to push the limits of this technology. In certain applications, CRISPR does enable generation of a model much faster compared to using embry- onic stem (ES) cell-based methods. Yet, CRISPR isn't necessarily the best approach for every project. Taconic Biosciences' experience indicates that CRISPR works extremely well for generating simple allelic configurations such as constitutive knockouts and knock in of point mutations, ac- cording to Adriano Flora, Ph.D., associate director, scientific pro- gram management, at the company. "However, it is not as well suited to introducing more com- plex modifications relying on homologous recombination over larger regions," he says. "While Taconic and others have done the latter successfully, the complexity of the effort can create longer and unpredictable timelines and higher costs, potentially ne- gating the reasons for selecting CRISPR in the first place." Dictating Genetic Modifications The balance of CRISPR's advantages with its limitations dic- tates the kind of genetic modifications that can be introduced and yields a subset of research projects for which the technol- ogy is most appropriate for developing a suitable animal model. An interesting application is model refitting: the introduc- tion of additional genetic modifications to well-established existing models where multiple modified alleles are already present, or the modification of transgenic alleles in already es- tablished mouse lines. "The latter application is ideal for testing the efficacy of a therapy on models that carry different variations of a human- ized gene," says Dr. Flora. "For example, Taconic has used CRISPR to develop mouse models with varying metabolizing levels of a human liver gene involved in drug metabolism, mimicking natural variants present in the human population." In the final analysis, successful use of CRISPR to generate ani- mal models demands proper evaluation of the specific research objective and project requirements, followed by selection of the most appropriate tool from the model generation toolbox. n Examining FnCas9 To perform genetic modification on a mouse strain using CRISPR, single guide RNA(s), Cas9 mRNA or protein, and a targeting vector (depending on the nature of the desired mutation) are microinjected into pronuclear- stage embryos. After genotyping/sequencing analysis, the founder mice are backcrossed to mice of the appropriate background strain to generate mice that carry the mutations. Founder mice are often mosaic (as shown by the multicolored mouse). As a result of this mosaicism, it is not always easy to achieve germline transmission of a particular mutant allele. However, once the allele has reached the germline, the allele will be transmitted by Mendelian genetics, as expected. The Jackson Laboratory

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