Genetic Engineering & Biotechnology News

MAY1 2015

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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20 | MAY 1, 2015 | | Genetic Engineering & Biotechnology News The simplest way to create a knockout model is to inject CRISPR/Cas reagents, in- cluding Cas9 mRNA and a single gRNA, into the mouse embryo. If a knockin alteration is small, the intended mutation can be accom- modated into a donor oligonucleotide of the maximal size of 200 bps; for larger altera- tions that cannot be engineered into a donor oligonucleotide, such as incorporation of a reporter gene or a human sequence, a donor plasmid is often used. "We are now exploring the use of CRISPR for larger-scale genetic manipulation and hu- manization of the mouse genome. We do not know yet the size limitation of the genetic manipulation that you can introduce with the CRISPR/Cas technology," discussed Wenning Qin, Ph.D., associate director of genetic engi- neering technologies, The Jackson Laboratory. The Jackson Laboratory uses insertion of a fuorescent reporter gene into the Nanog locus, a gene expressed in early embryos, as the plat- form for parameter optimization. To determine if there were any off-target effects in addition to the on-target insertion of the reporter gene into the Nanog locus, two HDR mice carrying the reporter gene were genome sequenced, and evaluated minimally for the top 5,000 sites. No off-target effects were observed indicating that the CRISPR/Cas reagent used had a clean off- target profle among the examined sites. Various means of enhancing the on-target effciency of CRISPR/Cas9 modifcations are being investigated by Taconic Biosciences. For example, the company is investigating the im- plementation of Cas9-orthologs to gain more fexibility in the choice of target sequences. It is also evaluating alternative delivery methods as well as experimental design and execution optimization. To date, analyses have focused on evalu- ating on-target modifcations. Nonetheless, various approaches to enhance specifcity— for example, the use of Cas9 nickase, trun- cated sgRNAs, and Cas9 protein instead of mRNA—are being assessed. According to Jo- chen Welcker, Ph.D., senior manager of scien- tifc development, improving the generation of more challenging types of alleles is a major development objective, and effciency issues for specifc applications need to be resolved. CRISPR Continued from page 18 OMICS The Cas9 (CRISPR associated protein 9) system has gained signifcant interest due to its relative simplicity and ease of use compared to other genome-engineering technologies, according to many scientists. The CRISPR/Cas9 system requires a complex of the Cas9 protein with a trans-activat- ing RNA (tracrRNA) and a gene-targeting CRISPR RNA (crRNA) or a single guide RNA (sgRNA, a chi- meric form of tracrRNA with a crRNA). Researchers at Dharmacon, now part of GE Healthcare, recently carried out a study on the efciency of using synthetic crRNA and tracrRNA to introduce gene-editing events when co-trans- fected with a plasmid expressing Cas9. They ex- plored the use of antibiotic and FACS methods for enrichment of cells that have undergone gene editing, and the use of multiple promoters to in- crease efciency of gene editing with Cas9 and synthetic tracrRNA and crRNA. The researchers reported that utilizing a highly active promoter for Cas9 expression en- ables better editing in specifc cell lines. Enrich- ment of transiently transfected cells either by fuorescence-activated cell soring or puromycin selection can further improve the yield of edited cells, they added. In addition, they concluded that efcient gene editing can be achieved with a three- component system: plasmid Cas9 and synthetic tracrRNA and crRNA. They also pointed out that use of synthetic tracrRNA and crRNA is a simpli- fed method for gene editing of one or more genes without requiring any cloning steps, and that the three-component CRISPR/Cas9 system is amenable to high-throughput genome edit- ing applications. n Gene Editing with Cas 9 and Optimal Promoter Th e Ja c k s o n L a b o rat o r y a d v i s e s t h at t h e simplest way to create a knockout mouse model is to inject CRISPR/Cas reagents, including Cas9 mRNA and a single gRNA, into a mouse embryo.

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