Genetic Engineering & Biotechnology News

MAY1 2015

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Genetic Engineering & Biotechnology News | GENengnews.com | MAY 1, 2015 | 21 For the generation of conditional knockout alleles, one obstacle is the effcient and correct integration of loxP-sites. Using ssODNs (single- stranded oligodeoxynucleotides) as donor mate- rial, for example, frequently leads to incomplete integration of the loxP-sites encoded by these ssODNs. The high effciency of NHEJ-mediat- ed deletion of the genomic region between the two loxP-sites is also a major hurdle as it leads to knockout alleles in up to 30% of the injected zygotes. The generation of complex knockin alleles is limited by the frequency of insertion/ replacement of longer sequences by HDR. Taconic has generated more than 200 hu- manized models by HDR. A drawback of this approach is the fact that only a single allelic variant can be modeled, while ever increasing amounts of human gene variants are being identifed by genome-wide association studies (GWAS). Such variants can now be introduced quickly and effciently, directly on existing hu- manized backgrounds by the use of CRISPR/ Cas9 genome editing, making it possible to generate a whole range of human allelic vari- ants from a single humanized-mouse model. Bioinformatics Needs "Bioinformatics plays an important role in CRISPR advancement. No-charge informatics packages are available, such as eCRISP, MIT tool, Doench activity scoring, and Zift, that tackle a portion of the informatics aspects to CRISPR design, and many commercial com- panies have simple design tools built into their reagent-ordering systems," stated Eric Rhodes, chief technology offcer, Horizon Discovery. "Our new tool, gUIDEbook, a collaboration between Horizon and DesktopGenetics, is the frst free application to combine all three as- pects of informed design." First, all available protospacer adjacent mo- tif (PAM) sites in a given region, which repre- sent a potential gRNA design, must be found. Virtually all CRISPR design tools enable this and differ primarily in how the sequence is entered and user interface complexity. All pro- grams essentially return the same information. Second, off-target cutting potential by any given gRNA must be determined. Historically, searches found closely related sequences and scores were generated using weighting based on the number of mismatches, their location in the gRNA, and the number of occurrences in the genome. A new fnding has demonstrated that "bulges" can occur in the matching of a gRNA to a potential target. More intensive searching is now required to identify all puta- tive binding sites. The unknown is how likely an off-target identifed by either method is ac- tually going to be engaged. The Doench algorithm, which is still early in its development, focuses solely on how much cutting activity a given gRNA design is likely to have. The algorithm calculates pre- dictive scores on the basis of known guides and cutting activities. It does not account for off-target potential, so it has somewhat lim- ited standalone value. Delivery Thermo Fisher Scientifc supplies a com- plete workfow for gene editing and cell en- gineering that focuses on design, delivery, and analysis. Transfection-grade Cas9 protein and mRNA have been functionally tested in several cell lines, including iPS and ES cells, and both contain a nuclear local- ization signal (NLS) to aid in delivery. The GeneArt Cas9 Nuclease is extensively puri- fed and quality controlled to remove nonspe- cifc endonucleases and endotoxins. According to Jason Potter, senior scientist of protein engineering, cell lines vary in how easily they can be transfected. With plasmids and mRNA, the cell must still process the transcripts and make Cas9 complexes before it can act. To simplify the process, the gRNA can be made and complexed with the trans- fection-grade Cas9 protein in vitro. After it is delivered by lipids or electroporation is used, the Cas9 complex is able to act once it reach- es the nucleus. Analysis of the edited cells can then be done using the GeneArt Genomic Cleavage Detection kit or by sequencing. Lipids, including Lipofectamine 3000, Li- pofectamine RNAiMAX, and Lipofectamine MessengerMAX, have been used for deliv- ery of plasmids and RNAs for years. Drawing on this knowledge of Cas9, the company has optimized lipid dosages and protocols for high transfection effciency and low toxicity. Due to the exposed guide RNA component of the Cas9 complex, RNAiMAX also works for de- livery of Cas9 protein. For electroporation, the key consideration is optimizing the voltage and pulse conditions for the cell line. CREATING TOMORROW'S SOLUTIONS 2.0 THE FAB EXPRESSION TECHNOLOGY ESETEC ® 2.0: READY FOR A NEW EXPRESSION? Are you looking for a new expression technology for the production of proteins and antibody fragments? With amazing yields? And marvelous cost ef ciency? Here it is: ESETEC ® 2.0. The advanced version of our patented E. coli secretion technology more than triples our record yields and reliably produces even complex molecules such as antibody fragments – at a productivity that exceeds industry-optimized processes for mammalian cell cultures (CHO cells). For more information, visit www.wacker.com/biologics Wacker Biotech GmbH, Jena/Halle, Germany, Tel. +49 3641 5348 - 206, info.biologics@wacker.com OMICS

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