Genetic Engineering & Biotechnology News

NOV15 2017

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16 | NOVEMBER 15, 2017 | | Genetic Engineering & Biotechnology News genes—and are also offered in predefined catalog collections or custom cherry-pick builds. The approach incorporates chemically modified synthetic guide RNAs (gRNAs) that provide a two-part guide RNA system simi- lar to the endogenous CRISPR/Cas9 mecha- nism. The two-part gRNA is more practical and cost-effective for high-throughput chemi- cal synthesis, so thousands can be made in a week—and scale up to larger amounts is much more straightforward. Four crRNAs per gene offer high statisti- cal power and a straightforward hit-ranking system. The crRNAs can also be pooled into a single reagent for screening applications. A validated gRNA selection algorithm ensures functionality and specificity (Figure 1). Best practices for CRISPR screening in- clude reconfirming hits following the primary screen by repeating the experiment on a small- er scale to highlight both weak and strong hits, and using different gRNAs per gene to determine if similar phenotypes result. Next is ensuring that the phenotype correlates with the genomic-level alteration, typically tested with a PCR-based mismatch detection assay. "We also recommend going back to RNAi to see how well gene silencing corre- sponds to gene knockout. Although the phe- notype may be slightly different, if RNAi re- sults agree with CRISPR results, it provides confirmation that the results are due to loss of function," explains Louise Baskin, senior product manager at Dharmacon. The company is also working on synthet- ic and lentiviral tools for CRISPR activation (CRISPRa), the activation of transcription by gRNAs that target sequences near the transcriptional start site. Lentivirus can transduce many relevant cell lines including nondividing cells. The Invitrogen LentiArray CRISPR Libraries also target the whole human genome (Figure 2). The library consists of Cas9 and gRNA particles along with positive and negative controls. Gene sets or custom picks are available as well. "gRNAs are designed to primarily 5 cod- ing exons of a target gene using our CRIS- PR gRNA design tool in order to maximize knockout efficiency and to minimize off-target effects. We have four highly ranking guides per gene; cleavage efficiency averages around 60% or greater for a more robust knockout and reproducible phenotype," discussed Jona- than Chesnut, Ph.D., senior director, synthetic biology R&D, Thermo Fisher Scientific. "Lentiviral titers are guaranteed to at least 1 x 10 6 TU/mL, and are tested using an antibiotic resistance assay to count actual in- fectious particles." Internal experiments, with a CellSensor (Thermo Fisher Scientific) line containing a reporter system driven by an NF-ᴋB tran- scription factor response element, looked at the effects of knocking out kinases known to be active in that pathway and showed very low assay-to-assay variability. 3 In another ar- rayed full-kinome screen, hits from a CRIS- PR and an siRNA knockdown screen were compared. 3 siRNA picked up the main play- ers; however, the CRISPR screen identified a dozen other known involved genes. Simone Treiger Sredni, M.D., Ph.D., as- sociate professor, Pediatric Neurosurgery, Northwestern University Feinberg School of Medicine, Ann and Robert H. Lurie Chil- dren's Hospital of Chicago, used a subset of the human kinome library for her work on malignant rhabdoid tumors, deadly embryo- nal tumors that occur in infants and toddlers. The tumors are associated with poor surviv- al outcomes and typically don't respond well to currently available therapies. 4 Dr. Sredni and colleagues mutated 160 kinases in a well-characterized rhabdoid tu- mor cell line using the kinome library sub- set, and then the investigators measured the clones' growth rates measured over a period of months. In 2–3% of the cell lines, growth rate slowed significantly. Further work deter- mined that the mutation of Polo-like kinase 4 (PLK4) by CRISPR/Cas9 produced a high- er impairment of cell proliferation, opening up the door for possible treatment options. Synthetic Single-Guide RNAs More research is needed to fully understand how toxic CRISPR components can be to liv- ing cells, and the type of immune responses they may elicit in the different therapeutic ap- proaches—such as human in vivo, human ex vivo, human germline, or antiviral- and antimi- crobial-based therapies. In addition, the likeli- hood of off-target effects needs to be thorough- ly assessed; some off-targets may be averted by using transient CRISPR therapeutics. As CRISPR evolves, gRNAs have evolved. Two-piece complexes consisting of crRNA CRISPR Tools Continued from page 14 OMICS Figure 2. Thermo Fisher Scientific is applying the power of the CRISPR/Cas9 system to high- throughput screening applications with the Invitrogen LentiArray CRISPR Libraries. The arrayed libraries of gRNAs are constructed in lentiviral expression vectors, which allows researchers to perform high-throughput screens in a wide variety of cell types. © 2017 MaxCyte, Inc. All Rights Reserved. Accelerating Your Discovery, Development and Manufacturing of Cell-based Medicines From concept to care, our delivery platform for cell engineering helps you harness the power of the cell. • The only cell engineering solution that scales from bench to bedside • Non-viral delivery platform for immuno- oncology, gene editing and regenerative medicine applications • Generate cell therapies with enhanced potency and efficacy • Increase safety with minimal toxicity • Clear regulatory pathway for streamlined development • 40+ partnered programs — making cell therapy a reality Any Cell. Any Molecule. Any Scale. ® Harness the power:

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