Genomic Editing & Screening

Sloan-Kettering Inst Can Research, New York NY

Investigators

Linked publications, trials & patents

Abstract

ABSTRACT The major goals of the Genomic Editing and Screening (GES) Core Facility are 1) to provide CRISPR-Cas9 sgRNA gene knockout and knock-in services targeting mutations/genes-of-interest towards the creation of stable cell lines, including adoption of base editing and prime editing techniques to enhance CRISPR knock-in efficiency for targeted edits and a lower risk of spurious indels; 2) to offer customized focused and full-genome knockout / knockdown technologies via pooled CRISPR-Cas9 and arrayed siRNA libraries, respectively; 3) to provide cutting-edge RNA interference (RNAi) gene target knockdown technologies to facilitate the discovery of novel cancer targets and complex, aberrant intracellular pathways, utilizing Pol II-driven, enhanced Mir-E-based shRNA for high-efficiency protein knockdown with single-copy integration using a panel of lentiviral plasmid backbones for delivery; 4) to provide small barcoding libraries (5-20K guides), such as CellTag (V1,V2, & V3), and large barcoding libraries (245K guides and greater), such as LARRY and Watermelon, for lineage tracing and subsequent delineation of lineage-associated behavior; 5) to provide the CRISPR-mediated transcriptional activation/interference (CRISPRa/CRISPRi) technologies to allow scientists to investigate induced/suppressed gene expression within native cellular context, therefore facilitating powerful applications in large-scale of gain-offunction/ loss-of-function genomic screens and the engineering of cell-based models; 6) to produce highly efficient lentiviruses in two formats (standard and concentrated) for transducing patient-derived xenograft (PDX) and cell lines. 7) to validate the efficiency of CRISPR and RNAi using western blotting (WB), real-time PCR (qPCR), and Immunofluorescence (IF) technologies; 8) to assist researchers in employing the Core’s high content phenotypic screening (HCS) platforms using fixed cells, including the Yokogawa CellVoyager CV8000 Nipkow spinning disk confocal HCS System, equipped with machine-learning functionalities as well as two GE Incell 6000 laser line scanning confocal imagers, equipped with a robotic arm plate-loader for screen automation; 9) to enable live cell imaging and kinetic experiments with the two Sartorius-Essen IncuCyte S3 systems, with modules for organoid tracking, cell-by-cell analysis, and advanced classification of live-cell phenotypes; 10) to support researchers with liquid handling workstation-based small molecule compound screening, allowing investigators to employ high throughput screening (HTS) chemical biology approaches with our collection of ~123,000 compounds. Promising compounds can then be matriculated into the Early Therapeutic Center (ETC) and Tri-Institutional therapeutic Drug Initiative (TRI-I TDI). Further, in the coming year the GES Core seeks to identify on-site research partners to launch a new initiative in Functional Precision Medicine (FPM), utilizing our HTS expertise and extensive chemical library collection to provide fPM services for clinicians and patients at MSKCC. Overall, this comprehensive Core Facility dedicated to functional genomics and screening enables mechanismbased science through target identification and phenotypic imaging, offering custom reagents, screening of small molecule compound collections, and cell line generation services to a large number of the MSK Center’s members.