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May 01, 2017 (Vol. 37, No. 9)

More Tooth, More Tail in CRISPR Operations

The Most Interesting Thing about CRISPR/Cas9 Is What It Can Accomplish in the Hands of Gifted Researchers

  • Pooled CRISPR Libraries

    Highly complex libraries of oligonucleotides, 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 synthesis, 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 increased 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, diagnostics and genomics division, Agilent Technologies. “The ability to generate libraries with uniform, even representation is directly tied to the spatial control and accuracy of the DNA printers.”

    A process derived from inkjet printing is used to deposit nucleotides to a growing chain on a solid support. Miniature jets contain the different dNTPs (nucleoside triphosphates containing deoxyribose), other monomers, 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 generate a plasmid library that maintains or improves the quality of the synthesized library, and then move downstream to begin addressing specific questions. In addition, a library-cloning 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 techniques, and will continue to evolve and change as needed.

  • CRISPR Mouse Models

    In the late 1980s, pronuclear injection 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 undesired 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-assisted 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 Laboratory. “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 immunodeficiency (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; backcrossing 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 currently working on understanding how to titrate gRNA/Cas9 activity and to make the process more efficient. For example, efficiencies could be realized though techniques such as batch electroporation, which can introduce reagents into embryos.

  • Knowing When CRISPR Is the Right Approach to Generate Your Model

    Click Image To Enlarge +
    To perform genetic modification on a mouse strain using CRISPR, single guide RNA(s), Cas9 mRNA or protein, and a targeting vector (depending on the nature of the desired mutation) are microinjected into pronuclear stage embryos. After genotyping/sequencing analysis, the founder mice are backcrossed to mice of the appropriate background strain to generate mice that carry the mutations. Founder mice are often mosaic (as shown by the multicolored mouse). As a result of this mosaicism, it is not always easy to achieve germline transmission of a particular mutant allele. However, once the allele has been transmitted through the germline, the allele will be transmitted as expected by Mendelian genetics. [The Jackson Laboratory]

    Demand for obtaining genetically engineered animal models on faster timelines has fueled the development of gene-editing technologies like CRISPR. As interest skyrockets, it is critical to evaluate how and where to employ CRISPR in the development of animal models for in vivo studies.

    The simplicity and efficiency of CRISPR, and the promise of shorter lead times, have led investigators to push the limits of this technology. In certain applications, CRISPR does enable generation of a model much faster compared to using embryonic stem (ES) cell-based methods. Yet, CRISPR isn’t necessarily the best approach for every project.

    Taconic Bioscience’s experience indicates that CRISPR works extremely well for generating simple allelic configurations such as constitutive knockouts and knock in of point mutations, according to Adriano Flora, Ph.D., associate director, scientific program management, at the company.

    “However, it is not as well suited to introducing more complex modifications relying on homologous recombination over larger regions,” he says. “While Taconic and others have done the latter successfully, the complexity of the effort can create longer and unpredictable timelines and higher costs, potentially negating the reasons for selecting CRISPR in the first place.”

    Dictating Genetic Modifications

    The balance of CRISPR’s advantages with its limitations dictates the kind of genetic modifications that can be introduced and yields a subset of research projects for which the technology is most appropriate for developing a suitable animal model.

    An interesting application is model refitting: the introduction of additional genetic modifications to well-established existing models where multiple modified alleles are already present, or the modification of transgenic alleles in already established mouse lines.

    “The latter application is ideal for testing the efficacy of a therapy on models that carry different variations of a humanized gene,” says Dr. Flora. “For example, Taconic has used CRISPR to develop mouse models with varying metabolizing levels of a human liver gene involved in drug metabolism, mimicking natural variants present in the human population.”

    In the final analysis, successful use of CRISPR to generate animal models demands proper evaluation of the specific research objective and project requirements, followed by selection of the most appropriate tool from the model generation toolbox. 

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