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Cellecta offers both pre-made and custom pooled lentiviral libraries for both CRISPRa targeted gene activation and CRISPRi targeted gene repression screens. These modified CRISPR platforms provide alternative genetic screening approaches that enable idenfication of genes whose modulation--either repression or activation--initiates a response.
Engineered versions of deactivated Cas9 (dCas9) have proven to be effective modulators of gene expression when complexed with repressor (e.g., dCas9-KRAB) or activator (e.g., dCas9-VPH) proteins and targeted to the upstream promoter regions of gene targets with sgRNA. This technology forms the basis of the “CRISPR interference” (CRISPRi) and CRISPR activation (CRISPRa) systems.
Cellecta’s CRISPRa and CRISPRi human genome-wide sgRNA libraries have been designed to target almost 19,000 human protein-coding genes using 5 sgRNAs designed against the promoter region based on Horlbeck, et al. (eLife 2016;5:e19760 DOI: 10.7554). Our dual-guide CRISPRa and CRISPRi libraries use the same 5 effective sgRNA in dual combination on each of 5 constructs (sgRNA 1 & 2, sgRNA 1 & 3, sgRNA 2 & 3, etc.) Libraries are available in 200 ug plasmid and pre-packaged 2 x 10^8 TU and 1 x 10^9 TU lentiviral particles.
Alternatively, for more targeted or specialized sets of genes, we can make a custom library to suit your requirements.
Two CRISPRa sgRNA to the Same Target Increase Expression. Two different sgRNA targeting the MyoD gene were transduced separately and together in triplicate into MDA231 cells. One of the guides activated expression poorly by itself, and the other induced a bit over 10-fold activation. However, when the both guides were combined in the same cell, overall induction increased by almost an additional 10-fold.
Researchers have engineered the Cas9 protein to eliminate the nuclease activity and produce a deactivated Cas9 (dCas9) that still binds tightly to the sgRNA so it can be targeted to essentially any location in the genome. When the dCas9 protein is fused a gene repressor protein (i.e., the KRAB protein) it forms a complex that can inhibit gene expression (CRISPRi) when targeted to the upstream region of a target gene. Conversely, gene transcription activators (e.g., VP16) can linked to the sgRNA/dCas9 to make a complex that can be targeted and activate a gene of choice. Since the effect does not alter the underlying DNA structure, like a CRISPR knockout does, the induction or repression is reversible.
Cellecta offers pre-made libraries targeting all protein-coding genes for genome-wide CRISPRa and CRISPRi screen in both human and mouse systems. Custom libraries can also be build that target any genes sets of interest, as well as non-coding RNA and regulatory domains.
Pooled libraries of sgRNA in conjunction with dCas9 activator or repressor complexes enable broad based gene screens that assess the effects of activation or repression on thousands of different genes on cell growth or other screenable phenotypes. The CRISPRa system, in particular, is the only approach that enables genome-wide gain-of-function screens to identify endogenous genes that change phenotypes when activated, rather than disrupted.
Like the standard CRISPR knockout system, CRISPRa and CRISPRi pooled libraries screens can be used to identify potential therapeutic targets responsible for controlling growth and differentiation, regulating disease development, or other biological responses. The development of the new dual-sgRNA CRISPRa and CRISPRi libraries extends the range of tools available for functional genetic screening and accelerates the identification of novel targets for therapeutics and biomarker analysis.
