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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14 | DECEMBER 2017 | GENengnews.com Arrayed Library Screening The arrayed synthetic form of CRISPR/Cas9 allows many differ- ent assays (e.g., enzymatic, endpoint, secreted factors) to be performed without resorting to antibiotic selec- tion, long time points, or cell splitting, procedures that may be required with the vector-based single-guide RNA (sgRNA) approach, explains Dr. Vermeulen. "These additional procedures can cause noise for certain readouts, so expression of sgRNA in arrayed for- mat can be difficult to implement in a high-throughput setting." Predesigned or custom synthetic guide RNAs can be ordered on Dhar- macon's website. "Ordering these predesigned reagents greatly simplifies the process of doing a CRISPR/Cas9 experiment," advises Dr. Vermeulen. "One need not be an expert in the design aspects of CRISPR/Cas9 tech- nology to complete experiments using this technique. Being able to transfect the cells with a lipid reagent or electro- poration becomes the only technical requirement." The Dharmacom technology supports several experimental ap- proaches. "For example," notes Dr. Vermeulen, "our system could be used to knockout a gene or genes in a cell line and then assay the cell line with a chemical library of small compounds in a high-throughput screen." Compared to RNA interference (RNAi) systems, such as those making use of short hairpin RNA (shRNA), short interfering RNA (siRNA), and double-stranded RNA (dsRNA), CRISPR editing permanently modifies genomic DNA rather than transiently modulating RNA expression levels. The ability to perform permanent gene knockout experiments, in addition to transcription knockdown, provides researchers a more complete under- standing of gene function. Library Generation Another technical advance is the ability to generate synthetic DNA that has much more integrity than the syn- thetic DNA that was generated in the past. "We are now able to synthesize DNA with an error rate equal to or better than the current fidelity of next- generation sequencing," asserts Benja- min Borgo, Ph.D., global senior prod- uct manager, Agilent Technologies. He explains that the company lev- eraged this increase in DNA fidelity to synthesize ultra-high-quality CRISPR libraries, adding that this enhance- ment in DNA quality leads to a corre- sponding improvement in the quality of many of the company's molecular biology and genomics reagents that rely on synthetic DNA as an input. "Another exciting advance is the length of DNA we can synthesize, which has increased alongside our improvements in fidelity. People are often amazed to hear that Agilent has the ability to print an entire human genome in a single run on one of our DNA writers," he says. The solutions that Agilent Genome Engineering: CRISPR Proving More User-Friendly Continued from page 12 B E S T O F C R I S P R 2 017 ➜ CRISPR technology has revolutionized the process for creating genetically modified mice, allowing for shorter timelines and, in many cases, more efficient production. In a presentation entitled, "Utilizing CRISPR Technology to Develop Mouse Models of Human Disease," at the Hanson Wade CRISPR 2017 Conference, David S. Grass, Ph.D., senior director of genetic engineering, genotyping, and reproductive sciences at The Jackson Laboratory (JAX), discussed the impact of CRISPR technology on the JAX Model Generation platform. Dr. Grass highlighted the ability and experience of the JAX team to create genetic modifications on a wide array of genetic backgrounds (important for creating changes on strains that have preclinical relevance) and contrasted the CRISPR technol- ogy with more traditional techniques, such as homologous re- combination in mouse embryonic stem (mES) cells, for creating genetic modifications. "CRISPR technology increases the efficiency of homologous recombination such that the genetic modification can be per- formed in single-cell embryos, as opposed to mES cells," said Dr. Grass. "In addition to decreasing the timelines and the cost for creating the genetically modified mice, this allows for the work to be performed directly on different genetic backgrounds for which robust mES cells don't exist, avoiding extensive backcross- ing from the strain the mutation was made on to the preferred background strain." Case studies were presented for two projects performed for Cat Lutz, Ph.D., an investigator at JAX. For one, an allelic series was created for familial amyotrophic lateral sclerosis (FALS) us- ing an oligo-mediated knockin strategy to create different single nucleotide polymorphisms in the endogenous TUBA4 gene that are similar to those associated with patients with FALS. The other project involved the creation of a model for Fried- reich's ataxia, for which a conditional Frataxin null allele was cre- ated using a double-stranded DNA plasmid-mediated knockin strategy. The detailed design strategies for both projects were reviewed during the meeting. The processes for validating the mice also were discussed. These include polymerase chain reaction and sequence analysis, designed to ensure that the desired mutation exists in the ge- nome and has occurred at the targeted locus. n Genetically Modified Mice

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