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Cas9 Expression Vectors for More Efficient Genome Editing

Cas9 Nuclease and Nickase Expression Vectors
The level of Cas9 endonuclease expression has been shown to affect the frequency of generating genome-edited clones. The efficiency of delivery and resulting expression of the targeting components vary widely between cell types.  Even within a given cell population, nuclease expression levels often vary significantly.  Consequently, only a small fraction of cells within culture express sufficient levels of Cas9 to facilitate double-strand break activity.

Duda et al 2014 showed that selection of high expressing Cas9 cells (using GFP or other markers as a fluorescent readout) resulted in a 4 to 20- fold increase in genome editing efficiency. Figure 1 shows the direct correlation between Cas9 expression level and indel frequency.

Figure 1. Fluorescent markers provide a method of enrichment for cells with increased indel frequency.    GFP expression was directly linked to Cas9 expression via 2A peptide.  FACS was used to bin cells into low medium and high expression of the fluorescent marker (and Cas9).  Indel frequency was measured using the CEL-I Surveyor assay and the percentages are shown at the bottom of each lane.  Cells enriched for the highest fluorescence expression showed the highest indel frequency.


Highlights:

  • Transfect or transduce target cell lines with lentiviral Cas9 expression vectors- Transduction allows for efficient generation of Cas9 stable cell lines.
  • Multiple Selectable markers and Reporters - Blasticidin and/or puromycin to perform antibiotic selection selecting for Cas9 expressing cells. Co-selection can be performed with gRNA expression vectors for more efficient indel production. Fluorescent reporters  (ZsGreen or tRFP) in Cas9 vectors allow for FACS sorting of high expressing Cas9 cells.
  • Inducible Cas9 expression allows the creation of cell lines that limits constitutive expression of Cas9 which may reduce off target effects and may also be a necessary component when knockout of an essential gene is being considered.
  • Creation of stable cell lines with Cas9 or inducible Cas9 allows for more uniform expression of Cas9 which is desirable in the context of larger multiplexed screens.
  • Paired gRNA with Cas9 Nickase  for gene editing - Use Cas9 D10A mutant expressing vectors in combination with 2 gRNAs to produce efficient and specific gene editing (Ran et al., 2013).

Vector Specifications:

transEDIT™ Cas9 Nuclease expression vectors incorporate selection strategies to select for high Cas9 expressing cells and variable promoters to optimize expression in target cell lines. Tet-inducible Cas9 vectors have been developed to allow for more efficient control over Cas9 expression in your target cell line.

Cas9 expression vectors
(pCLIP-Cas9 Nuclease, pCLIP-Cas9 Nickase)

transEDIT CRISPR Cas9 Nuclease Expression Vectors

Cat# Product Name Literature Price Buy
TECC1001 transEDIT CRISPR Cas9 Nuclease Expression Vector (pCLIP-Cas9-Nuclease-EFS-Puro) View Request Pricing Contact Distributor
TECC1002 transEDIT CRISPR Cas9 Nuclease Expression Vector (pCLIP-Cas9-Nuclease-EFS-Blast) View Request Pricing Contact Distributor
TECC1003 transEDIT CRISPR Cas9 Nuclease Expression Vector (pCLIP-Cas9-Nuclease-EFS-ZsGreen) View Request Pricing Contact Distributor
TECC1004 transEDIT CRISPR Cas9 Nuclease Expression Vector (pCLIP-Cas9-Nuclease-EFS-tRFP) View Request Pricing Contact Distributor
TECC1005 transEDIT CRISPR Cas9 Nuclease Expression Vector (pCLIP-Cas9-Nuclease-hCMV-Puro) View Request Pricing Contact Distributor
TECC1006 transEDIT CRISPR Cas9 Nuclease Expression Vector (pCLIP-Cas9-Nuclease-hCMV-Blast) View Request Pricing Contact Distributor
TECC1007 transEDIT CRISPR Cas9 Nuclease Expression Vector (pCLIP-Cas9-Nuclease-hCMV-ZsGreen) View Request Pricing Contact Distributor
TECC1008 transEDIT CRISPR Cas9 Nuclease Expression Vector (pCLIP-Cas9-Nuclease-hCMV-tRFP) View Request Pricing Contact Distributor

transEDIT CRISPR Cas9 Nickase Expression Vectors

Cat# Product Name Literature Price Buy
TECN1001 transEDIT CRISPR Cas9 Nickase Expression Vector (pCLIP-Cas9-Nickase-EFS-Puro) View Request Pricing Contact Distributor
TECN1002 transEDIT CRISPR Cas9 Nickase Expression Vector (pCLIP-Cas9-Nickase-EFS-Blast) View Request Pricing Contact Distributor
TECN1003 transEDIT CRISPR Cas9 Nickase Expression Vector (pCLIP-Cas9-Nickase-EFS-ZsGreen) View Request Pricing Contact Distributor
TECN1004 transEDIT CRISPR Cas9 Nickase Expression Vector (pCLIP-Cas9-Nickase-EFS-tRFP) View Request Pricing Contact Distributor