Genetic Engineering & Biotechnology News

DEC 2017

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32 | DECEMBER 2017 | GENengnews.com B E S T O F C R I S P R 2 017 Genome -Wide CRISPR Screen Identifies New Class of Regulatory Proteins Donato Tedesco, Ph.D., Director of R&D, and Paul Diehl, Ph.D., COO, Cellecta U sing a CRISPR-based genetic screen, Cellecta recently identified, for a client, a class of proteins responsible for regulating gene expression of a genetic element present in the 5′-untranslated region (5′-UTR) of a gene involved in a specific disease indication. For this study, we introduced Cellecta's 80K Genome- Wide CRISPR Knockout Library, which expresses CRISPR single guide RNAs (sgRNAs) targeting all known human protein-coding genes (four sgRNAs/gene), into two isogenic cell lines. Both cell lines were engineered with a green fluo- rescent protein (GFP) expressed from the same promoter. However, one cell line had the genetic regulatory ele- ment of interest inserted between the promoter and GFP reporter, and the other did not. At a few different time points, then, both cell lines were sorted by flow cytometry to separate the cells that brightly fluoresced from those with low fluorescence (Figure ). The relative abundance of cells transduced with each sgRNA in the populations of high- vs. low-fluorescent cells col- lected at each time point was assessed by high-throughput sequencing. This analysis revealed a number of sgRNAs over- represented in the low-fluorescing population that had the genetic element of interest, as opposed to the control cells that did not. These sgRNAs targeted a small group of genes that appear to regulate expression of this element since their disruption reduced expression of the GFP reporter. In conclusion, with this one assay we were able to screen all human protein-coding genes and identify a set of proteins responsible for controlling this therapeutically interesting genetic regulator. CRISPR/Cas9 Arrayed Screening with Synthetic crRNA in Primary T Cells as a Platform to Accelerate HIV Therapy Development Louise Baskin Senior Product Manager, Dharmacon, a Horizon Discovery Group Company F or years, high-throughput arrayed functional genomics screening has been a standard for understanding biological pathways. The ability to analyze phenotypes on a one-well-per- gene basis supports sophisticated phenotypes, including high-content assays, and enables a richer investigation of biological mechanisms than a pooled screen. There are hundreds of examples of these loss-of-function screens with RNAi, but only recently have we seen arrayed studies with CRISPR/Cas9-mediated gene knockout. A 2016 publication (J.F. Hultquist et al., "A Cas9 Ribo- nucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells," Cell Rep. 17(5), 1438–1452 [2016]) elegantly demonstrates the power of arrayed CRISPR screening using the Dharma- con Edit-R™ synthetic guide RNA platform. Their screen leveraged a robust, high-throughput assay system that includes electroporation of CRISPR/Cas9 ribonucleopro- teins (RNPs) consisting of Cas9 protein with a synthetic CRISPR RNA (crRNA) and tracrRNA guide RNA. The study was focused on identifying novel targets that impact HIV infectivity. As a proof of concept, the re- searchers performed a high-throughput CRISPR screen of 146 different RNP complexes targeting 45 genes involved in HIV integrase function. In a 96-well format, RNPs were electroporated into primary T cells isolated from several different blood donor samples. The robustness of this approach was validated by measurement of phenotype, gene-editing percentage, and gene knockout. The authors were able to confirm expected phenotypes when knocking out several host factors and also discovered new gene targets. Thus, they demonstrated the usefulness of a scalable, arrayed CRIS- PR knockout screening approach to identify novel host factors for HIV pathogenesis and infection. This platform is expected to have utility in elucidating other biological pathways in primary T cells as well as other primary cells important in human disease. Applications of Novel CRISPR Tools Figure. Cellecta's CRISPR technology enables genetic screens and the development of genome-edited cell lines.

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