The Functional Genomics Core facility provides infrastructure for unbiased Target Discovery by means of genome-wide genetic screening in cultured cells. The Core’s primary effort is dedicated to loss-of-function screening via RNAi and CRISPR-CAS9 technologies although other services such as micro-RNA (miR) screening and cell population barcoding for clonal analysis are also available. Our range of expertise covers from project feasibility assessment, all the way through verification of identified targets. In addition to screens, the Core produces CRISPR-engineered cell lines to serve as models for screening or to aid the follow-up process, and can provide reagents tailored to the needs of each project such as sh/siRNAs, ORF clones, CRISPR reagent aliquots. Our personnel develop both plate reader and image-based cellular assays, and adapt them to high-throughput screening in arrayed format in conjunction with the High Content Screening Core. CRISPR-CAS9 screens are performed in pooled format using either off-the shelf or custom made lentiviral libraries, and results are de-convoluted in our Genome Analysis shared resource.
Consultation: Provide expert advice on all steps in a functional genomics experiment, from initial design to execution of high throughput projects.
RNAi and miR screening (384 well format; plate reader and HCS assays)
- Assay development: cellular assays are taken from the researcher, tested under transfection/ transduction conditions, and brought to a 384-well format. Z’ factor is assessed to determine the feasibility of the project.
- Screen execution: siRNA/lentiviral collections are screened in 384-well format in pools of 4 siRNAs per gene. Following QC, raw data are statistically analyzed and results are returned to the investigator for hit selection. Screens are performed in duplicate, although there is the option of adding more data points for an additional fee.
Hit cherry-picking and confirmation: selected hits are cherry-picked as 4 single siRNAs per gene and plated in 384-format. Up to 16 hit collection copies are provided to allow a re-run through the primary assay as well as through additional secondary assays. Results are analyzed and returned to the investigator.
CRISPR-CAS9 Knock-out screening (viability/resistance assays)
- CAS9 cell line construction: cells are transduced with either constitutive or DOX inducible CAS9 lentiviral vectors. Individual clones are tested for optimal cleavage activity and the 2 best clones are selected for screening.
- Assay development: the CAS9 line is expanded and tested in large culture format. sgRNAs targeting essential genes or controls are used to estimate optimal timeline of the experiment. Best Compound concentration and background resistance parameters are evaluated.
Screen execution: the screen is performed in triplicates targeting 300 cells per sgRNA in the initial transduction step. After the treatment takes its course, cells are harvested, DNA extracted and libraries amplified, indexed and QCed for sequencing. Sequencing is then performed in a NextSeq sequencer. Results are delivered as frequencies per sgRNA construct.
Cell line engineering/genome editing services
Flag tag insertion, either N terminal or C-terminal
Luciferase reporter insertion
Cell population barcoding services: High diversity lentiviral libraries carrying millions of barcodes can be transduced into cells to create cell populations where each cell is recognizable by a single barcode which is passed to daughter cells, allowing lineage tracing for purposes such as tracking the dynamics of heterogeneous populations. We provide the barcoded cell line of choice. The service includes QC-ing via NGS to document the diversity and homogeneity of the barcoded line.
Support/ custom reagents procurement
siRNA, shRNA or ORF clone distribution: We will provide any siRNA, miR or lentiviral clone present in our libraries at affordable cost.
Custom shRNA set construction and validation: We generate a set of 6 lentiviral-shRNAs (either constitutive or inducible) against any given target and provide 2 ml of viral supernatant of each construct. For an additional fee we will create shRNA cell lines and determine silencing efficiency by QPCR.
CAS9 cell line construction: either constitutive or DOX inducible CAS9 expression. For an additional fee we will deliver it transduced with any of our libraries
GeCKO v2 library, viral supernatants: transduction-ready library preps in separate sets each containing 3 sgRNAs/gene. each aliquot is sufficient
The following libraries are available for screening and clone distribution:
- siRNA libraries:
• Human genome-wide ON-TARGET-PLUS (OTP, Dharmacon, 18301 targets)
• Human ubiquitinome library (OTP, Dharmacon, 1170 targets)
• Human kinome library (OTP, Dharmacon, 659 targets)
• Human phosphatase library (OTP, Dharmacon, 202 targets)
• Human cancer pathways set (OTP, Dharmacon, 252 targets)
• Custom siRNA libraries, either pooled or as single siRNAs.
- Lentiviral shRNA libraries:
• Human Kinome Lenti-shRNA library (TRC, SIGMA). Targets 672 genes, averaging 11 shRNA constructs per gene.
- miR libraries:
• Human miRVANA miRNA mimic library (Life Technologies, targets 2565 miRs)
• Human miRCURY LNA™ miR-family inhibitor library (Exiqon, targets 43 miR families)
• Human miRCURY LNA™ microRNA Inhibitor library (Exiqon, targets 1972 miRs)
- ORF libraries (gain-of-function screening):
• Expression-ready lentiviral secreted protein library (Life Technologies, 858 clones)
• Other libraries, such as kinases, apoptosis/survival and innate immunity gene sets are also available
- CRISPR-CAS9 pooled libraries
• Human CRISPR Knockout Pooled Library (GeCKO v2), targets 19050 genes with 6sgRNAs per gene. Available as 1 or 2 vector system
• Human Activity-Optimized CRISPR Knockout Library subsets (Lander/Sabatini library)
• Control library (100 non targeting sgRNA constructs
• Nuclear proteins (3733 targets)
• Cell Cycle Proteins (983 targets)
• Kinases (507 targets)
• Proteins of Unknown Function (1808 targets)
- Cell Barcoding libraries:
• Lentiviral ClonTracer Barcoding Library
- Benchcell work station with Bravo liquid handling platform (Agilent)
- STAR liquid handling station (Hamilton)
- Tissue culture facility
- WellMate liquid dispenser (Matrix Technologies)
- Micro Flo Liquid Dispenser (BioTek)
- ELx 405 Plate washer (BioTek)
- High-Throughput Microscopes and Plate Readers are utilized via the High Throughput Screening and High Content Screening
For a Price List, please call (858)646-3100 ext. 3537 or email us.
Pedro Aza-Blanc, Ph.D.
Dr Aza-Blanc earned his Ph.D. degree in the Universidad Autonoma de Madrid in 1994 working in the field of Molecular Endocrinology. In 1995, he moved to San Francisco where he trained in developmental biology at the University of California-San Francisco. In 1999, he joined the Genomics Institute of Novartis in San Diego as a Principal Investigator in the Genomics Department, where he performed pioneering work in such as the development of the first algorithms for improved siRNA performance, the creation of the first siRNA libraries, and the application of high-throughput RNAi screening technology. His work resulted in the first publication describing RNAi screening as a discovery tool in mammalian systems. He was recruited to SBP in 2006 to launch the Functional Genomics Core, where he currently serves as Director. Altogether, Dr. Aza-Blanc brings more than 12 years of experience on functional genomics and target discovery both in academic and industrial environments.
Please call (858)646-3100 ext. 3537 or use the button below to send us an email.