Mouse CRISPR Activation Library (SAM - 3 plasmid system) Pooled Library #1000000075 BioVector NTCC中国质粒载体菌种细胞基因保藏中心

Mouse CRISPR Activation Library (SAM - 3 plasmid system) Pooled Library #1000000075

描述：

SAM协同激活介导因子(synergistic activation mediator) CRISPR-Cas9系统质粒激活内源基因转录表达

 CRISPR/Cas9 Synergistic Activation Mediator (SAM) is an engineered protein complex for the transcriptional activation of endogenous genes. It consists of three components:

1.A nucleolytically inactive Cas9-VP64 fusion,

2.An sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop 2

3.The MS2-P65-HSF1 activation helper protein. SAM can be combined with a human genome-wide library to activate all known coding isoforms form the RefSeq database (23,430 isoforms) for gain-of-function screening.Library description: The SAM library consists of 3 unique sgRNAs targeting each human RefSeq coding isoform in the proximal promoter (> 90% of sgRNAs are targeted to the first 200bp upstream of the TSS of their target). The total library size is 70,290 guides. For SAM gain-of-function screening, this sgRNA library has to be combined with two additional SAM constructs – dCas9-VP64 and MS2-P65-HSF1. These are provided along with the library.

Screening:After amplification of the library DNA according to the provided protocol, lentivirus is produced for the library, dCas9-VP64 and MS2-P65-HSF1 separately. The design of the three lentiviral vectors is shown below:

The Lentivirus of all three components is then titrated and target cells are transduced with dCas9-VP64 and MS2-P65-HSF1 at MOI <1 selected="selected">500 cells per guide in the library. This equals >175 million cells for the initial transduction and maintenance of > 35 million cells after completed selection with Zeocin. After 7 days of selection with Zeocin, cells are ready for screening to begin.

Cells have to be transduced with all three SAM components.

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