Edit-R Synthetic Positive crRNA Controls

Synthetic crRNA controls to verify optimal parameters for CRISPR-Cas9 gene editing


Positive control crRNA for confirming gene editing within your experimental conditions, provided with or without primers for your DNA mismatch detection assay of choice.

Edit-R Synthetic crRNA Controls are designed and validated for mismatch detection assays to verify gene editing experiments. They are recommended as positive controls for CRIPSR-Cas9 experiments utilizing synthetic crRNA to optimize transfection conditions and verify Cas9 nuclease expression.

Controls are configured as follows:

  1. Individual Edit-R crRNA Control for human, mouse, or rat (5 nmol or 20 nmol) designed against:
    1. Cyclophilin B (PPIB)
    2. DNA (cytosine-5)-methyltransferase 3B (DNMT3B)

  2. Edit-R crRNA Control Kits for human. mouse, or rat containing the following:
    1. Either a 5 nmol or 20 nmol Edit-R CRISPR Synthetic crRNA Control
    2. 5 nmol Edit-R crRNA Control forward primer
    3. 5 nmol Edit-R crRNA Control reverse primer

Remember to order Edit-R tracrRNA for use with your controls!

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. When Cas9, the endonuclease component of a CRISPR-Cas system, is complexed with two RNAs called the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA), it forms a complex that cleaves DNA. This flexible system can be exploited to induce site-specific genome modifications to program, regulate and precisely interrogate gene function.

The Dharmacon Edit-R CRISPR-Cas9 platform includes the three critical components required for gene editing in mammalian cells, based on the natural S. pyogenes system:

  • plasmid or lentiviral particles expressing a mammalian codon-optimized gene sequence encoding Cas9 Nuclease
  • chemically synthesized trans-activating CRISPR RNA (tracrRNA), and
  • chemically synthesized CRISPR RNA (crRNA) designed to target the gene of interest

Once delivered into the cell, the crRNA: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 protein knockout.

How much crRNA & tracrRNA do I need?

This table provides the approximate number of experiments that can be carried out for lipid transfection methods at the recommended crRNA:tracrRNA working concentration (25 nM:25nM) in various plate/well formats. Calculations do not account for pipetting errors.
crRNA
nmol
tracrRNA
nmol
96-well plate
100 µL reaction volume
24-well plate
500 µL reaction volume
12-well plate
1000 µL reaction volume
6-well plate
2500 µL reaction volume
2 2 800 160 80 32
5 5 2000 400 200 80
10 10 4000 800 400 160
20 20 8000 1600 800 320
  
HazardousYes
Shelf Life12 Months
Shipping ConditionAmbient
Storage Condition-20 C
Less time preparing more time experimenting

Less time preparing more time experimenting

Less time preparing more time experimenting

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


PPIB positive controls with promoter selection.jpg

Effective gene editing of PPIB in human and mouse cells

PPIB positive controls with promoter selection.jpg

A human recombinant U2OS ubiquitin-EGFP proteasome cell line (Ubi[G76V]-EGFP) (A) and a mouse fibroblast (NIH/3T3) (B), were stably transduced with lentiviral particles containing Cas9 and a blasticidin resistance gene driven by the indicated promoters.. A population of cells with stably integrated Cas9-blastR was selected with blasticidin for a minimum of 10 days before transfections. Cells were transfected with 50 nM synthetic crRNA:tracrRNA targeting Human PPIB / mouse Ppib using DharmaFECT 1 and DharmaFECT 3 Transfection reagent, respectively. After 72 hours, the relative frequency of gene editing was calculated based on a DNA mismatch detection assay using T7EI on genomic DNA extracted from the transfected cells.


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. L. Cong, F. A. Ran, et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 339(6121), 819-823 (2013).
  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).
  14. R. Barrangou, A. Birmingham, et. al. Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference. Nucleic Acids Research43(7) 3407-3419 (2015)

Application Notes

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