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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Page 60 of 69 | DECEMBER 2017 | 27 screen for one gene, a couple, or the whole genome." The arrayed library will continue to evolve. Dialing a gene up or down is looking plausible, and the libraries may be developed for use in gene acti- vation and repression. Pooled CRISPR Libraries Highly complex libraries of oligo- nucleotides, including pooled CRISPR libraries, require the synthesis of hundreds to hundreds of thousands of unique oligos in a single library. When produced using a traditional, column- based approach, each individual oligo needs to be pooled following synthe- sis, opening the door to errors. Column-based synthesis is not the best approach for pooled CRISPR libraries. With recent developments, Agilent's synthesis fidelity has in- creased significantly with the ability to synthesize libraries in which it is difficult to differentiate errors from synthesis versus sequencing errors. The throughput of the platform combined with tight spatial control allows active tuning of biases within every library. Both the chemistry and the process to print DNA have been refined, and biases are measured using methods such as DNA sequencing. "The quality difference is apparent in many of our oligo-based products but most recently in our catalog and custom SureGuide pooled CRISPR libraries," stated Ben Borgo, Ph.D., global senior product manager, diag- nostics and genomics division, Agilent Technologies. "The ability to generate libraries with uniform, even representa- tion is directly tied to the spatial control and accuracy of the DNA printers." A process derived from inkjet print- ing is used to deposit nucleotides to a growing chain on a solid support. Miniature jets contain the different dNTPs (nucleoside triphosphates containing deoxyribose), other mono- mers, and chemicals. These nozzles place droplets of reagent into specific sites on the solid support. A generalized workflow allows researchers to design their own library or utilize Agilent's designs, synthesize the library quickly, inexpensively gen- erate a plasmid library that maintains or improves the quality of the synthe- sized library, and then move down- stream to begin addressing specific questions. In addition, a library-clon- ing technique that eliminates the need for agar plates has been developed. As new Cas9 variants are being described, each application requires a new library. Libraries have already been developed for some of these tech- niques, and will continue to evolve and change as needed. CRISPR Mouse Models In the late 1980s, pronuclear in- jection was first performed to make transgenic mice. The ability to modify an endogenous gene in the mouse genome came later. It was achieved by homologous recombination in mouse embryonic stem (mES) cells that were injected into blastocyst embryos to create chimeric mice. These chimeric mice could be bred to produce mice derived solely from the mES cells. This inefficient process requires selection, often leaves behind unde- sired genetic material, and relies on skilled mES cell culturing so that they retain pluripotency and can populate the germline. Initially, mES cells were isolated from the 129 mouse strains. More recently, they were isolated from other stains. Creating genetically modified mice on other background strains requires a long backcross breeding process. "With CRISPR and nuclease-as- sisted homologous recombination we can go directly into the embryo and create the desired mutation directly on many mouse inbred strains," discussed David Grass, Ph.D., senior director, genetic engineering, The Jackson Lab- oratory. "In addition to performing projects on C57BL/6J and BALB/cJ, we have even been able to work in the immune-deficient NSG mice. "NSG mice carry two mutations including severe combined immunode- ficiency (SCID) known to be involved in DNA repair. So we were not sure how efficient the CRISPR would be. But it has worked really well, allowing us to work on the 'next generation' of humanized mice." It used to take 1–1.5 years to produce a germline mouse; backcross- ing took another 1–1.5 years. With CRISPR, it takes 6–8 months. From a cost perspective, for simple projects, such as small indels, CRISPR is 81% less costly; for the more complicated homologous recombinations, it is 40% less costly. Fortunately, if off-target events occur in mice, they can be segregated from the desired allele when the mice are bred, unless the off-target is closely linked in the genome to the desired mutation. The Jackson Laboratory is cur- rently working on understanding how to titrate gRNA/Cas9 activity and to make the process more efficient. For example, efficiencies could be real- ized though techniques such as batch electroporation, which can introduce reagents into embryos. B E S T O F C R I S P R 2 017

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