Edit-R Cas9 Nuclease Expression Reagents

Non-lentiviral Cas9 expression plasmids with your choice of promoter and enrichment method


Edit-R Cas9 Nuclease Expression Reagents
Endotoxin-free, purified DNA for direct co-transfection with Edit-R synthetic crRNA and tracrRNA. Choose from three options to facilitate enrichment of Cas9-expressing cells.

Edit-R CRISPR-Cas9 Gene Engineering Platform

The Dharmacon Edit-R Gene Engineering platform is based on the Type II CRISPR-Cas9 system from the bacteria Streptococcus pyogenes which can be engineered and adapted to edit genes in mammalian cells. All of the Edit-R Cas9 Nuclease Expression plasmids encode a human codon-optimized version of the S. pyogenes Cas9 (Csn1) gene under the control of an RNA pol II promoter. Edit-R Cas9 Nuclease Expression plasmids are non-lentiviral vectors provided as endotoxin-free purified DNA for direct co-transfection with Edit-R synthetic crRNA and tracrRNA.

The recommended Dharmacon products for use with Cas9 Nuclease Expression plasmid are:

Once delivered to the cell, the crRNA and tracrRNA complex with Cas9 nuclease to generate site-specific, DNA double-strand breaks (DSBs). When DSBs are repaired through non-homologous end-joining (NHEJ), the resulting small insertions and deletions (indels) can cause nonsense mutations resulting in gene disruption to produce a functional knockout.

Highlights

There are three Edit-R Cas9 Nuclease Expression plasmid options from which to choose, based on preference of enrichment method and cell types:

  1. Edit-R Cas9 Expression plasmids with mKate2: Cas9 nuclease and the mKate2 fluorescent reporter are both expressed under the control of a single RNA pol II promoter, making this plasmid useful for downstream cell enrichment by FACS. Offered with your choice of six different promoters.
  2. Edit-R Cas9 Expression plasmids with PuroR: Cas9 nuclease and the Puromycin-resistance marker are both expressed under the control of a single RNA pol II promoter, making this plasmid useful for downstream cell enrichment by antibiotic (Puromycin) selection. Offered with your choice of six different promoters.
  3. Edit-R Cas9 Nuclease Expression plasmid with hCMV-BlastR: Cas9 nuclease expression is driven from a human cytomegalovirus (hCMV) promoter, and Blasticidin resistance (BlastR) is under the control of the Simian virus 40 (SV40) promoter. This simple vector is useful for those who do not want a fluorescent protein constitutively expressed in the cells of interest and prefer to enrich for Cas9-expressing cells through Blasticidin treatment, especially if a longer antibiotic selection time is required.

Not all RNA pol II promoters are equally active in different cellular environments

The activity of any given promoter controlling the transcription of Cas9 nuclease can differ greatly from one biological context to another, resulting in variable Cas9 expression levels and thus varying levels of DNA cleavage. Choosing an optimal promoter for your cell line or type will therefore affect the degree of gene editing in your experimentation. The Edit-R Cas9 Nuclease expression plasmids are offered with six different, well-characterized cellular promoters from which you can choose.

Six SMARTchoice promoter options for expressing Cas9 nuclease
Promoter Description
hCMV human cytomegalovirus immediate early promoter
mCMV mouse cytomegalovirus immediate early promoter
hEF1α human elongation factor 1 alpha promoter
mEF1α mouse elongation factor 1 alpha promoter
PGK mouse phosphoglycerate kinase promoter
CAG chicken beta actin hybrid promoter
  
HazardousNo
Shipping ConditionAmbient
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-20 C
Cas9 Nuclease Selection Guide

Which Cas9 nuclease is right for you?

Cas9 Nuclease Selection Guide

While the best Cas9 nuclease product for your experiment may heavily depend on the particular application or cell type, a few basic questions may help to point you in the right direction for product selection.


Workflow image comparing sgRNA cloning to Edit-R gene engineering.

Less time preparing, more time experimenting

Workflow image comparing sgRNA cloning to Edit-R gene engineering.

The Edit-R Gene Engineering system utilizes high-quality synthetic tracrRNA and crRNA to program Cas9 nuclease, thereby eliminating the need to clone individual sgRNAs thus saving time and labor.


Gene knockout workflow using Cas9 Expression plasmid and Edit-R crRNA:tracrRNA

Gene knockout workflow using Cas9 Expression plasmid and Edit-R crRNA:tracrRNA

Gene knockout workflow using Cas9 Expression plasmid and Edit-R crRNA:tracrRNA

An illustration of the Edit-R CRISPR-Cas9 components required for gene editing:plasmid expressing Cas9 nuclease, tracrRNA, and crRNA designed to the target site of interest. All three components are co-transfected into the mammalian cell of choice using DharmaFECT Duo Transfection Reagent to perform gene disruption. Enrichment of transfected cells can then be carried out by fluorescent cell sorting or selection for Puromycin resistance.


Vector elements and promoter options of Edit-R Cas9-mKate2 Expression plasmid

Vector elements and promoter options of Edit-R Cas9-mKate2 Expression plasmid

Vector elements and promoter options of Edit-R Cas9-mKate2 Expression plasmid

The Edit-R Cas9-mKate2 plasmid expresses the monomeric red fluorescent protein mKate2 and the human codon-optimized Cas9 nuclease from S. pyogenes, driven by one of six choices of RNA pol II promoters (Figure 2). By linking expression of mKate2 to Cas9 nuclease using the self-cleaving peptide T2A, sorting mKate2-positive cells by FACS will enrich for Cas9-expressing cells and increase the percentage of cells which have undergone the gene editing event.


Vector elements and promoter options of Cas9-PuroR Expression Plasmid

Vector elements and promoter options of Cas9-PuroR Expression Plasmid

Vector elements and promoter options of Cas9-PuroR Expression Plasmid

The Edit-R Cas9-PuroR plasmid expresses the Puromycin-resistance selection marker and the human codon-optimized Cas9 nuclease from S. pyogenes, driven by one of six choices of RNA pol II promoters (Figure 3). By linking expression of the Puromycin-resistance marker to Cas9 nuclease using the self-cleaving peptide T2A, selecting cells by treatment with puromycin will enrich for Cas9-expressing cells and thus increase the percentage of cells which have undergone the gene editing event.


Enrichment of Cas9-expressing U2OS cells using SMARTCas9-mKate2 expression plasmid by FACS results in increased target gene editing

Enrichment of Cas9-expressing U2OS cells using SMARTCas9-mKate2 expression plasmid by FACS results in increased target gene editing

Enrichment of Cas9-expressing U2OS cells using SMARTCas9-mKate2 expression plasmid by FACS results in increased target gene editing

Enrichment of Cas9-expressing U2OS cells using SMARTCas9-mKate2 expression plasmid by FACS results in increased % editing of human PPIB. U2OS cells were transfected with SMARTCas9-mKate2 (with human CMV promoter) expression plasmid and tracrRNA:crRNA targeting the human PPIB gene. Cells were sorted at 72 hours on a MoFlo XDP 100 instrument into three bins corresponding to negative, low, and high mKate2 fluorescence. SURVEYOR™ DNA mismatch assay was performed on sorted U2OS cells and % gene editing was compared with the unsorted (US) and control untransfected (UT) cells. The level of editing was calculated using densitometry (% editing). An increase in % gene editing is observed in the sorted cells, correlating with the increased mKate2 expression.


References

  1. D. Bhaya, M. Davison, et al. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu. Rev. Genet. 45, 273-297 (2011).
  2. L. Cong, F. A. Ran, et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 339(6121), 819-823 (2013).
  3. E. Deltcheva, K. Chylinski, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 471(7340), 602-607 (2011).
  4. Y. Fu, J. D. Sander, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. (2014).
  5. D.Y. Guschin, A. J. Waite, et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol.
    Biol. 649, 247-256 (2010).
  6. F. Heigwer, G. Kerr, et al. E-CRISP: fast CRISPR target site identification. Nat. Methods. 11(2), 122-123 (2014).
  7. P.D. Hsu, D. A. Scott, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31(9), 827-832 (2013).
  8. M. Jinek, K. Chylinski, et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 337(6096), 816-821 (2012).
  9. P. Mali, L. Yang, et al. RNA-guided human genome engineering via Cas9. Science. 339(6121), 823-826 (2013).
  10. N. K. Pyzocha, F. A. Ran, et al. RNA-Guided Genome Editing of Mammalian Cells. Methods Mol. Biol. 1114, 269-277 (2014).
  11. D. Reyon, C. Khayter, et al. Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REALFast assembly. Curr. Protoc. Mol. Biol. 100, 12.15.1‐12.15.14 (2012).
  12. T. R. Sampson, D. S. Weiss. Exploiting CRISPR/Cas systems for biotechnology. Bioessays. 36(1), 34-38 (2014).
  13. T. Wang, J. J. Wei, et al. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 343(6166), 80-84 (2014).