You are here: 分子生物学サービス » 人工遺伝子合成 » GenCRISPR™ gRNA構築 » SAM library for human genome-wide transcriptional activation
GenScript Share

SAM libraries induce robust transcriptional activation for genome-wide gain-of-function screening

SAM libraries induce robust transcriptional activation for genome-wide gain-of-function screening

The SAM library enables gain-of-function screening in human cells by inducing robust, specific transcriptional activation of each coding region in the genome in a single screen. CRISPR/Cas9 Synergistic Activation Mediator (SAM) is a protein complex engineered to enable robust transcriptional activation of endogenous genes. SAM takes advantage of the specificity and ease of reprogramming of Cas9 nucleases, which are targeted to a specific locus in the genome by guide RNA. The SAM complex includes three transcription activation domains – VP64, P65, and HSF1 – which synergistically enhance transcription when targeted to any site within 200bp upstream of a transcription start site. SAM uses a nucleolytically inactive Cas9 to ensure that no strand breaks are introduced into endogenous genome. A human genome-wide SAM library activates all known coding isoforms in the RefSeq database (23,430 isoforms) for gain-of-function screening. SAM libraries have been used in primary mouse and human cells, stem cells, cancer cells, and stable cell lines. SAM library screens allow you to identify genes that are essential for cell viability under different conditions, as well as genes whose loss enables drug resistance in cancer cells.

For complete details of SAM library design & construction, please see: Konermann S*, Brigham MD*, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O & Zhang F. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature, doi:10.1038/nature14136.

How to use SAM libraries for gain-of-function screens

The SAM library is a mixed pool of CRISPR guide RNAs that target immediately upstream of every transcription start site in the genome. For SAM gain-of-function screening, the human genome-wide SAM sgRNA library has to be combined with two additional SAM constructs – dCas9-VP64 and MS2-P65-HSF1. Cells must be transduced with all three SAM components. To enable co-transduction and selection, all three lentiviral vectors have unique resistance markers. All three components are delivered in lentiviral vectors optimized to produce high-titer virus for efficient lentiviral transduction of primary cells or cultured cell lines. A cell population should be transduced with the SAM library pool at a low MOI ensuring no more than 1 gRNA enters any given cell. After transduction, deep sequencing with NGS should be performed to assess gRNA representation in the cell pool before beginning a screening protocol. At the end of the screen, a second round of NGS is performed, and data analysis should be performed to identify the guides that were lost or enriched over the course of the screen. In order to identify true positive hits from a SAM library screen, you should identify genes for which multiple guides were enriched. A more detailed SAM screening protocol may be found on the SAM / Genome Engineering website.

SAM Library Components

Three components -- dCas9-VP64, sgRNA-MS2, and MS2-p65-HSF1 – form the assembled SAM complex

The SAM complex consists of three components:

  • A nucleolytically inactive Cas9-VP64 fusion
  • An sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop
  • The MS2-P65-HSF1 activation helper protein

The incorporation of three distinct activation domains - VP64, P65 and HSF1 – into the complex aids robust transcriptional activation through synergy. Each of the three components are encoded on a separate lentiviral vector as described below. All three vectors must be co-transduced in order for SAM to be active

All SAM human genome-wide guide RNA library sequences are available in this .csv file.

SAM Library Vectors

GenScript delivers SAM libraries as mixed pools of sgRNA cloned into the guide RNA lentiviral expression vector, accompanied by the dCas9-VP64 and MS2-p65-HSF1 lentiviral expression vectors which must be co-delivered.

Vector Name Selection Marker Vector Map
lenti sgRNA(MS2)_zeo
Zeo
lenti dCas9-VP64_Blast
Blast
lenti MS2-P65-HSF1_Hygro
Hygro

Related Services for CRISPR/Cas9 genome editing

How to order SAM Libraries

Please inquire by emailing gene@genscript.com. GenScript generates amplified and validated SAM libraries according to the SAM library amplification protocol provided by the Feng Zhang laboratory, followed by next-generation sequencing to quantify guide RNA representation and pool complexity.

Legal Statement of GenCRISPR Services and Products (Updated on July 28, 2015):

  1. GenCRISPR™ services and products are covered under US 8,697,359, US 8,771,945, US 8,795,965, US 8,865,406, US 8,871,445, US 8,889,356, US 8,889,418, US 8,895,308, US 8,906,616 and foreign equivalents and licensed from Broad Institute, Inc. Cambridge, Massachusetts.
  2. .The products and the reagents generated from these services shall be used as tools for research purposes, and shall exclude (a) any human or clinical use, including, without limitation, any administration into humans or any diagnostic or prognostic use, (b) any human germline modification, including modifying the DNA of human embryos or human reproductive cells, (c) any in vivo veterinary or livestock use, or (d) the manufacture, distribution, importation, exportation, transportation, sale, offer for sale, marketing, promotion or other exploitation or use of, or as, a testing service, therapeutic or diagnostic for humans or animals.
  3. The purchase of the GenCRISPR Services and Products coveys to the purchaser the limited, non-transferable right to use the products purchased and the reagents generated from GenCRISPR services and any related material solely for Research Purposes only, not for any Commercial Purposes.

Have questions? GenScript's Ph.D.-level service representatives are available 24 hours a day, Monday through Friday, to assist you.


Email: japanmarket@genscript.com
TEL: 03-6803-0843
Fax:03-3836-0371, 03-4496-5000(受注専用)