Genetic Engineering & Biotechnology News

NOV15 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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Genetic Engineering & Biotechnology News | GENengnews.com | NOVEMBER 15, 2017 | 17 and trans-activating crRNA (tracrRNA) are based on the native bacterial system. Single guide RNAs (sgRNAs)—hybrid, single-mol- ecule guides developed several years ago— have also been shown to be accurate, repro- ducible, and stable. Before the availability of synthetic sgRNAs, many labs produced CRISPR guides using an approach known as in vitro transcription (IVT), whereby enzymes are used to convert a DNA template into an RNA molecule. This time-con- suming approach yields highly variable prod- ucts requiring significant purification. Recently, new technological advancements have allowed for the synthesis of full sgRNAs (Figure 3). "A significant advantage that the synthetic sgRNAs have over the IVT-derived sgRNAs is that they can be chemically modified to provide stability and protection to the guide. This has enabled extremely high editing ef- ficiencies in primary T cells and stem cells, where unprotected IVT-derived guides typi- cally fail. Now available at a practical scale, price, and turnaround time, synthetic sgRNA is likely to become the de facto standard for CRISPR gRNAs," said Kevin Holden, Ph.D., head of synthetic biology, Synthego. The first clinical trial utilizing CRISPR, aimed at programming immune cells to tar- get and kill lung cancer tumor cells, began in China in late 2016. Currently, most CRIS- PR trials are in the preclinical phase. Given that many of these therapeutic approaches will rely on a transient CRISPR system, it is highly likely that they will incorporate either purified Cas9 nuclease, or Cas9 mRNA and a synthetically generated sgRNA. Standardizing Custom Library Design Often, mixing disciplines can have a ben- eficial effect. Oxford Genetics was founded to bring a level of standardization to genetic engineering, an approach typically seen in the engineering sciences. The use of automation and scalable processes increase predictability and robustness while lowering cost and hu- man error. According to Ryan Cawood, Ph.D., chief executive officer, Oxford Genetics, the goal is to minimize steps in complex library de- sign to improve turnaround time. Individual pieces of the CRISPR process are locked down, validated individually (to ensure that they work independently), and then validat- ed in combination with other pieces. Libraries are built using high-throughput automation into a modular plug-and-play background, like a car chassis, and can be modified in a number of ways, such as dock- ing sites, expression systems, viral vectors, etc., providing extra flexibility if the research direction changes. Custom pooled and arrayed CRISPR librar- ies are are developed in client-led efforts. Pooled libraries can contain up to 50,000 gRNAs in a tube, and arrayed libraries can reach 1000 gRNAs in one well of a 96-well plate. Custom CRISPR screening services are expected to be automated in the next six months. The company focuses its R&D on adding economic value to the pharmaceutical devel- opment workflow: discovery, development, manufacturing, and delivery. One avenue is using CRISPR to research and reengineer vi- ruses for manufacturing use. One example is a reengineered virus that goes through its life cycle repressing itself from producing its own proteins and redirecting itself toward produc- ing the protein of interest. Other foci include using CRISPR knockouts to facilitate adeno- associated virus and lentivirus production. Although the company is not involved in therapeutic development, Dr. Cawood be- lieves that CRISPR will play a big part in the future of regenerative medicine. CRISPR, he says, will kick-start stem cells, reawaken the growth potential that exists within the body, and facilitate genetically engineered replace- ment organs. References available online. Learn how to accelerate your pilot-scale media manufacturing at bd.com/One-Stop *Non-GMP pilot production. Additional time for shipping. © 2017 BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company. MC8392 FOR SCALABLE, ONE-STOP CELL CULTURE MEDIA PRODUCTION, TURN TO BD. BD continually advances solutions to support process development and manufacturing for scientists. BD ™ Rapid Media Solutions delivers a 10-business-day * turnaround on developmental medium production. Each custom formulation is evaluated by our team of cell culture media development experts to ensure manufacturing suitability at both pilot- and full-scale production. For consistency, we develop every formulation as a hydratable-to-liquid powder in our full-service rapid media pilot facility, which replicates the equipment and processes of our large-scale media manufacturing plant. The result? A fast and reliable one-stop solution for every stage of media development from initial testing through clinical trials. Discover the difference of a faster turnaround time and full-service solution. Discover the difference of BD. OMICS Figure 3. This chart compares the editing efficiency (solid bars) and consistency (black lines) of Synthego's CRISPRevolution sgRNAs against in vitro transcribed guide (IVT) RNAs in HEK293T cells. The experiment was conducted by a third party using three different gene targets and was replicated three times.

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