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.
- siRNA library preparation and arrayed screen:
The core has genome-wide human siRNA library ON TARGET-Plus (OTP) library (18,301 genes, Dharmacon (Dh) / Thermo Scientific (TS)), formatted as SMARTpools and 4 single siRNAs per gene. Seventeen collections of pre-spotted pathway-focused libraries include (1) druggable genome (7740 genes, Applied-Biosystems (AB) / Life Technologies (LT)); (2) epigenetics / transcriptional-regulators (1362 genes, AB/LT); (3) epigenetics (882 genes, created by SBP in-house); (4) ubiquitinome (1170 genes, Qiagen and 1432 genes, SBP); (5) kinome (704 genes, AB/LT and 831 genes, SBP); (6) cancer pathway (252 genes, Dh/TS); (7) phosphatases (231 genes, Dh/TS and 314 genes, SBP); (8) G protein coupled-receptor (GPCR) (Dh/TS); (9) metabolome (160 genes, AB/LT); (10) miniscreen random subset (Dh/TS); (11) autophagy (1601 genes, SBP); (12) innate immunity (96 genes, SBP); (13) B cell activation (127 genes, SBP); (14) T cell activation (287 genes, SBP); (15) ion channel (380 genes, SBP); (16) apoptosis (823 genes, SBP). Customized siRNA cherry-pick service is available upon request. In addition, CRISPR/Cas9 arrayed screening with (17) kinome gRNA library (789 genes, TS) is available.
- miRNA mimic and antagonist library arrayed screen:
The core has human miRNA mimic library (875 miRNAs from miRBase sequence database Version 10.1., AB/LT) and miRVana library (2565 miRNAs, TS). Libraries of miRNA antagonist include human miRIDIAN miRNA inhibitor (885 anti-miRNAs from miRBase 13.0 release, Dh/TS), Exiqon miRCURY LNA™ Inhibitor and microRNA Family Inhibitor (Qiagen). Collections of pre-spotted autophagy pathway-focused libraries include miR mimics (259 mimics, SBP) against autophagy-related genes and miR antagonists (255 antagonists, SBP) targeting autophagy-related miRNAs.
- Lentiviral shRNA and open reading frame (ORF) overexpression library arrayed screen:
The core has RNAi Consortium (TRC) lentiviral kinome shRNA library (SIGMA) covering 672 kinases and regulators with an average of 11 shRNAs per target. 2440 sequence verified ORFs from the Ultimate ORF Collection (LT) are available as sub-libraries including secreted protein (858 ORFs), apoptosis (476 ORFs), kinase (373 ORFs), and innate immunity (172 ORFs).
- CRISPR/Cas genome-wide / sub- library preparation and pooled screen:
The core has CRISPR lentiviral pooled libraries including two human genome-wide (GW) knock-out (KO) (Root and Doench, Zhang labs), one mouse GW KO (Teichmann lab), three human GW activation (Root and Doench, Weissman, Zhang labs), and two human GW inhibition libraries (Root and Doench, Weissman labs). Human pathway-focused pooled libraries contain (1) CRISPR/Cas9 KO sub-libraries of kinases; cell cycle proteins; nuclear proteins; unknown function; control targets (Sabatini and Lander lab) and (2) CRISPR/dCas9 inhibition sub-libraries of kinases, phosphatases, and drug targets; stress and proteostasis; mitochondria, trafficking, and motility; gene expression; membrane proteins (Weissman lab) as well as (3) SBP unpublished CRISPR/Cas9&Cas12 KO sub-libraries of kinome; epigenetics; ubiquitome; autophagy; phosphatase; ion channel; cancer pathway; innate immunity developed in-house. In addition, the core offers custom pooled single, dual (Cas9), triple (Cas12a), and quadruple (3 Cas12 + 1 Cas9 or 4 Cas12) gRNA(s) library construction services.
- Custom CRISPR/Cas gene disruption and editing:
The core provides custom creation of isogenic cell lines including gene knock-out, mutation and repair, as well as reporter insertion. Approaches to express CRISPR include lentiviral transduction and transient transfection with in vitro transcribed (IVT) RNA. Moreover, custom genetic modified mouse creation is accomplished via Rosa26 loci knock-in on C57BL/6 C2 and 129 mESCs.
- High-activity CRISPR/Cas stable cell line enrichment:
To avoid clonal line screening, the core has developed multiple fluorescent reporters capable of bulk-sorting to enrich lentiviral-transduced cells with high-efficiency SpCas9 and EnAsCas12a for CRISPRko; SpnCas9-ABE, CBE and A&CBE for CRISPR DNA adenine (A) and cytosine (C) base-editing (BE); dCas9-effectors for CRISPRon/off.
- Custom gene knock-down and overexpression via CRISPR inhibition, activation, on and off:
Repurposing CRISPR/Cas9 for promoter/enhancer regulation allows any gene and particularly long non-coding RNA (lncRNA) to be (1) inhibited through CRISPRi-dCas9-Krab (Weissman lab) or CRISPRoff-dCas9-Krab-DNMT3 (Gilbert lab) and (2) activated via three available CRISPRa-dCas9-VP64 systems, including SAM (Zhang lab), suntag (Weissman lab), and split fluorescent protein (Huang lab) as well as CRISPRon-TETv4 (Gilbert lab).
- NGS-ready amplicon library preparation and quality control:
The core is equipped with Dual Index Primers Set (NEB) which is compatible with Illumina NGS platform and capable of multiplexing up to 96 pooled amplicon libraries. Multiple quality control procedures are integrated to ensure the library complexity and quality during the amplification.
- Automated mini-scale production:
The robotic high throughput mini-scale services include clonal cell line creation, mammalian genomic DNA extraction, plasmid DNA isolation, DNA quantification and normalization, as well as lenti-/retro-viral production in 96-well plate format.
- Cell population barcoding:
Retrievable barcode technology such as CloneTracer barcoding lentiviral pooled library (Stegmeier lab) is available to create individually barcoded cell populations for linage tracing. Monitoring the unique barcode and number of individual barcode provides a quantitative analysis to study the clonal origin and heterogeneity in cell proliferation, differentiation, as well as resistance to the drug treatment.
Equipment & Resources
- 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.