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CRISPRa and CRISPRi Human Genome-Wide Libraries

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. To enable CRISPRa or CRISPRi screens, Cellecta offers:

  • Pooled genome-wide lentiviral CRISPRa and CRISPRi libraries for screens targeting almost 19,000 human genes
  • Libraries which comprise two sgRNAs targeting each gene for stronger activation and repression (dual-sgRNA constructs library)
  • Both plasmid (200ug) and pre-packaged lentiviral particle (2x 10^8 TU & 1x 10^9 TU) formats available

 

CRISPRa targeted gene activation and CRISPRi targeted gene repression provide alternative genetic screening approaches to identify genes required to maintain a biological response or, with CRISPRa, genes whose activation initiates a response—a gain-of-function screen. Cellecta offers pre-made, pooled lentiviral libraries targeting all human protein-coding genes for both types of screens.

Dual sgRNA CRISPRa Library Activates More Genes

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.

Many gene activators and repressors complex with the promoter region of their target genes in multiple places. Cellecta has found that using this multiplex approach enhances the gene modulation effect, particularly with CRISPRa.

CRISPRa Single vs. Dual sgRNA to Same Gene

 

Increased Gene Activation with Dual-sgRNA. To detect whether the CRISPRa dual-sgRNA Library increases the levels of gene activation as compared to the single-sgRNA CRISPRa library, several hundred cells transduced with either the standard single sgRNA library or the dual-sgRNA library were sorted by FACS into small populations of ca. 10 cells/well. Both genomic DNA and RNA was isolated from each set of cells. The sgRNA library constructs in the cells were identified from the genomic DNA, and the DriverMap Targeted RNA-Sequencing Assay was used to assess the expression level of all human protein-coding genes. The data was then analyzed to correlate the expression of the targets for the sgRNAs identified in each cell group. The comparison showed that 33% of the gene targets were not induced with the single-sgRNA library, as compared with 27% with the dual-sgRNA CRISPRa Library, and, correspondingly, 7% more genes showed an induction of greater than 10-fold with the dual-sgRNA CRISPRa library.  

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 Horbeck, 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, 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.

Human Genome-Wide CRISPRa Single-sgRNA and Dual-sgRNA Libraries

Selective activation of targeted genes when used in cells expressing dCas9-VPH (or other dCas9-activator variations compatible with spCas9 sgRNA designs)

Human Genome-Wide CRISPRi Single-sgRNA and Dual-sgRNA Libraries

Selective expression inhibition of targeted genes when used in cells expressing dCas9-KRAB (or other deactivated Cas9 repressors compatible with spCas9 sgRNA designs)

 

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