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 66 of 69 | DECEMBER 2017 | 33 Overcoming Bottlenecks in Screening for Clones Kyle Luttgeharm, Ph.D., Application Scientist, AATI A s use of the CRISPR/Cas systems expands, new bottlenecks have emerged. A significant bottleneck is the screening for clones containing induced mutations and the determination of the zygosity. While sequencing can be used as a screening method, cost and time requirements prevent its widespread adoption. As a result, scientists are turning to low-cost screening steps to reduce the number of individuals and pools sequenced. Heteroduplex cleavage assays are an efficient meth- od to screen for CRISPR-induced modifications, while simultaneously allowing the determination of gene ed- iting frequency. Heating, then slowly cooling PCR prod- ucts allows the formation of heteroduplexes between wild-type and edited amplicons. After heteroduplex formation, the sample is treated with an endonuclease, such as T7 Endonuclease I, which recognizes and cleaves at DNA mismatches. Advanced Analytical Technologies, Inc. (AATI) has developed a robust T7 endonuclease assay (AccuCleave T 7 C E ) that seamlessly integrates with the Fragment Analyzer™ Automated CE System, allowing for accurate determination of CRISPR gene editing frequencies. To determine the zygosity, AATI developed statistical models based on cleavage ratios that allow researchers to charac- terize mutations made in monoclonal diploid cell lines or organisms. AATI has fully automated the CRISPR detection and quantification workflow, enabling rapid screening of edited cell lines, animals, and plants—putting crucial results in researchers' hands sooner. Alt-R S.p. HiFi Cas9 Nuclease 3NLS: Highly Specific Genome Editing, Even under Challenging Conditions Brian Wang, Ph.D., Genomic Tools Market Development Manager, Integrated DNA Technologies A n ongoing concern for many re- searchers is potential off-target effect introduced by the CRISPR/Cas9 system. Existing CRISPR guide RNA de- sign tools often fail to effectively predict off-target sites. Delivery of the genome-editing reagents as an RNP complex, reduces but does not eliminate off-target edit- ing. Some popular Cas9 mutants that provide improved specificity compromise on-target efficiency, resulting in poor utility when delivered in RNP format. The latest addition to the highly potent Alt-R CRISPR- Cas9 system—Alt-R Streptococcus pyogenes HiFi Cas9 Nuclease 3NLS—achieves a perfect balance between genome editing efficiency and accuracy. IDT scientists took a unique approach to evolve the Cas9 nuclease into a more precise genome-editing tool. By screening over 250,000 mutants, they discov- ered this enzyme effectively eliminates the risk of un- predictable off-target cleavage typically observed with the use of wild-type Cas9. Unlike rationally designed Cas9 variants that suffer from a moderate to severe loss of on-target activity, the Alt-R S.p. HiFi Cas9 Nuclease 3NLS preserves a high level of editing efficiency at intended on-target sites while radically reducing off-target effects. Its superior performance has been validated by a number of prominent laboratories conducting transla- tional research with various biological systems. The re- lease of this high-fidelity Cas9 enzyme is a significant step toward the therapeutic use of CRISPR. Type 1 Diabetes Mouse Model David S. Grass, Ph.D., Senior Director, Genetic Engineering, Transgenic Genotyping, and Reproductive Sciences, The Jackson Laboratory T he Jackson Laboratory's Model Generation Services (MGS) team has extensive experience using CRISPR technology to create genetically modified mice, not just in the C57BL/6J strain, but in a wide variety of inbred mouse strains. This capability is significant because when developing a new genetic model, consideration of background genetic effects is essential to the therapeu- tic relevance of the new strain. In one example, David Serreze, Ph.D., a principal inves- tigator at The Jackson Laboratory, was interested in test- ing the hypothesis that the Aicda gene, which is required for class switch recombination/somatic hypermutation in B lymphocytes, is important in the development of type 1 diabetes. He selected the NOD/ShiLtDvs inbred strain, a polygenic model for autoimmune type 1 diabetes. Creating the new strain directly on the NOD back- ground avoided time consuming and expensive back- crossing, while also significantly reducing the number of mice needed. The CRISPR-generated NOD.Aicda −/− mice were com- pared with the parental NOD strain. Type 1 diabetes development was decreased in the NOD.Aicda −/− mice, suggesting that class switching is required for the devel- opment of type 1 diabetes. Aicda is known to be a part of the same functional pathway as RAD51. NOD mice were dosed with B E S T O F C R I S P R 2 017 ➜

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