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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34 | DECEMBER 2017 | GENengnews.com the small molecule 4,4′-diisothiocyanatostilbene-2,2′- disulfonic acid, which inhibits RAD51-mediated homolo- gous recombination. Similar to NOD.Aicda −/− mice, type 1 diabetes development was significantly decreased in the treated mice. The creation of CRISPR/Cas9-driven modifications of the Aidca gene on the NOD background strain by The Jackson Laboratory's MGS team showed that the Aidca/ RAD51 functional pathway might be a target for develop- ing compounds that can block type 1 diabetes develop- ment. This work is potentially translatable to the clinic as an approach to block the development of type 1 diabetes. Transfection Technology Aids Development of CRISPR-Based Thera- pies for Multiple Monogenic Diseases Linhong Li, Ph.D., Director of Cell Engineering, MaxCyte C RISPR is a transformative technol- ogy at the crossroads of basic research, drug discovery, and human therapeutics. Whether for use in basic research or as a therapeutic agent, CRISPR gene-editing machinery must be delivered to the relevant cell populations. As research grows more sophisticated and studies move to the clinic, the delivery of CRISPR machinery— whether in the form of proteins, RNA, or DNA—be- comes challenging as the target cells are frequently more difficult-to-transfect cells, such as primary cells or induced pluripotent stem cells (iPSCs). MaxCyte offers an easy-to-use, highly flexible de- livery platform that is based on Flow Electroporation™ technology and that facilitates efficient gene editing. MaxCyte's platform is designed for high performance, reproducibility, and scalability, and it can provide a reg- ulatory pathway to support gene-editing applications ranging from R&D to global gene therapy. In 2014, MaxCyte joined collaborative research efforts to develop potentially curative CRISPR-based therapies for multiple monogenic diseases such as X-linked chron- ic granulomatous disease (X-CGD) and sickle-cell dis- ease (SCD). MaxCyte's partners in these efforts include Harry L. Malech, M.D., chief of the Genetic Immuno- therapy Section at the National Institute of Allergy and Disease, and John F. Tisdale, M.D., senior investigator in the Molecular and Clinical Hematology Section at the National Heart, Lung, and Blood Institute. Cells harvested from X-CGD or SCD patients were edit- ed ex vivo using a CRISPR-mediated nuclease system and MaxCyte's delivery platform. High transfection efficiencies and cell viability of patient cells provided for clinically rel- evant gene correction rates for both indications. In addition, short- and long-term in vivo engraftment models demonstrated the restoration of function to im- mune cells affected by X-CGD. (S.S. De Ravin et al., CRIS- PR/Cas9 Gene Repair of Hematopoietic Stem Cells from Patients with X-Linked Chronic Granulomatous Disease," Sci. Transl. Med. 9(372), (2017); S.S. De Ravin SS et al. Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat Biotechnol. 2016, 34(4): 424–9.) Both collaborations remain in progress and are focused on bringing these treatments to market and making curative gene editing therapies a reality. Expedite the Analysis of CRISPR/Cas9 Products by Automating the Measurement of Editing Efficiency Zhiyong Peng, Ph.D., Application Scientist, Applied Genomics, PerkinElmer U sing CRISPR/Cas9 to make precise gene modifications requires robust analytical tools to verify the success- ful generation of on-target mutations, and to measure the efficiency of the experiment during protocol optimiza- tion. Traditional CRISPR/Cas9 analysis is bottlenecked by the lack of an efficient analytical tool to confirm editing events in a large pool of samples. Classical analysis protocols are time consuming and not amenable to high-throughput analysis. Perki- nElmer's LabChip ® GX Touch™ Nucleic Acid Analyzer uses microfluidic electrophoresis to automate the qualitative and quantitative analysis of CRISPR/Cas9 modified prod- ucts. This reduces the number of samples that need to be confirmed by sequencing and increases the capacity of the CRISPR workflow. The LabChip GX Touch Nucleic Acid Analyzer offers real-time analysis of the presence of desired mutations in less than 60 seconds per sample. The mutations are identi- fied by electrophoretic mobility shifts of the heteroduplex strand, a signature peak of on-target gene editing. In com- bination with digestion of the heteroduplex strand with T7 Endonuclease I and analysis of the resulting fragments, the on-target efficiency of gene editing can be determined. The LabChip GX Touch Nucleic Acid Analyzer allows a pool of samples to be screened in a high-throughput fashion. The editing efficiency of up to 384 samples can be analyzed within a few hours, resulting in a stream- lined protocol for the analysis of CRISPR/Cas9 products. For research use only. Not for diagnostic procedures. B E S T O F C R I S P R 2 017

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