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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26 | DECEMBER 2017 | GENengnews.com working on CRISPR clinical applica- tions is CRISPR Therapeutics. This company's initial emphasis is on ex vivo indications. Ex vivo indications have the benefit of a facile delivery ap- proach, such as electroporation, and the ability to characterize the edits be- fore administering treated cells to the patient. Plus, measuring biomarkers to understand phenotypic effects in a relatively short timeframe after therapy administration is straightforward. The company's lead indication, a compound to treat inherited single- gene hemoglobinopathies (such as sickle cell and beta thalassemia), relies on gene disruption to upregulate fetal hemoglobin. This approach could be curative. A large number of studies show that patients who have sickle- cell or beta-thalassemia traits are asymptomatic when they have upregu- lated fetal hemoglobin. "We can achieve gene disruption today using CRISPR/Cas9 with rela- tively high efficiency, more than 80– 90%," asserted Sam Kulkarni, Ph.D., chief business officer, CRISPR Thera- peutics. "Gene correction approaches are being improved continuously, but the efficiency of correction is still in the 50–60% range for hematopoietic stem cells." The key challenges to overcome include delivery, pharmacology, and manufacturing. CRISPR/Cas9 is a multicomponent system and needs to be delivered to the target organs or tis- sues for in vivo applications. Previous work on small interfering RNA (siR- NA) and other therapeutic modalities may prove beneficial. Careful analysis is required to characterize the type of edits and the fraction of cells edited. This pharma- cology hurdle is easier to clear ex vivo than in vivo. Finally, manufacturing involves multiple components and also complex cell manufacturing. Collec- tive efforts of private and academic laboratories are rapidly surmounting these issues. Recent advances that result in high levels of homology-directed repair are facilitating efforts to expand the ad- dressable indications with CRISPR. Some approaches attempt to impact the level of cycling of the cells; others utilize modifications of the donor tem- plate and guides; and yet others are working on optimization of the pro- cess of CRISPR/Cas9 directed repair. Arrayed CRISPR Libraries Tools play an important role in making the prospect of high-through- put knockout screening a reality. Such tools have been pioneered by MilliporeSigma, which has launched various CRISPR products. The first such product consisted of simple constructs/plasmids that could accom- modate targeting elements and yield custom clones. Soon after developing this product, MilliporeSigma realized that a large collection of clones would be of great utility. A previous collaboration between MilliporeSigma and the Broad Insti- tute had resulted in a short hairpin RNA (shRNA) library, and the goal was to duplicate that model to de- velop a product that would work for the majority of researchers. Another collaboration, this one between MilliporeSigma and the Wellcome Trust Sanger Institute, had a similar vision, and after two years of work, it generated its first com- plete arrayed whole-genome CRISPR screening libraries. The off-the-shelf libraries offer substantial cost sav- ings and facilitate standardization of CRISPR screening. Although pooled libraries have been available for a while, the arrayed libraries provide one clone for one gene in one well, reducing ambiguity about the target at screen completion. The human and mouse libraries were designed to knock out virtually every protein-coding gene in their respective genomes, and each library contains two unique and highly specific gRNAs for every gene target. The sec- ond clone is used to verify that the first is not an artifact or a false positive. The human library contains ap- proximately 34,000 clones targeting 17,000 genes, and the mouse library contains approximately 40,000 clones targeting 20,000 genes. Bacterial glyc- erol stock, plasmid DNA, and lentivi- ral formats are available. "The beauty of the Sanger designs is that they will hit that gene and that gene only," commented Shawn Shafer, Ph.D., director, advanced genomics, MilliporeSigma. "Anything that did not meet our stringent design strategy did not make it into the library. "Some genes are tiny or highly repetitive, and so these genes were not suitable targets. Now you can Gifted Scientists Rapidly Advance CRISPR Operations Continued from page 25 B E S T O F C R I S P R 2 017 With CRISPR and nuclease-assisted homologous recombination, we can go directly into the embryo and create the desired mutation directly on many mouse inbred strains.

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