banner: Edit-R CRISPR-Cas9 Gene Engineering

Edit-R CRISPR-Cas9 Gene Engineering

The Dharmacon Edit-R™ CRISPR-Cas9 platform greatly simplifies the
workflow of permanently knocking out genes by eliminating the
time-consuming cloning of individual guide RNA expression vectors. Our
novel approach includes transfection-ready DNA and RNA components and
enables fast assessment of multiple target sites per gene, for multiple
genes.





Gene editing with the CRISPR-Cas9 system

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


  • a plasmid expressing a mammalian codon-optimized gene sequence encoding Cas9 nuclease
  • a long, chemically synthesized trans-activating CRISPR RNA (tracrRNA)
  • a chemically synthesized CRISPR RNA (crRNA) designed to cleave the gene target site of interest
edit-r Figure 1: Gene editing with the CRISPR-Cas9 system
Figure 1.
Illustration of Cas9 nuclease (1), programmed by the tracrRNA (2) :
crRNA (3) complex cutting both strands of genomic DNA 5' of the PAM (4).
All three components can be co-transfected into the mammalian cell of choice using the Dharmacon DharmaFECT™ Duo Transfection Reagent
to perform gene knockout. Once delivered to the cell, the crRNA and
tracrRNA complex with Cas9 nuclease to generate site-specific,
double-stranded DNA breaks (DSBs). When DNA DSBs are repaired through
non-homologous end-joining (NHEJ), the resulting small insertions and
deletions (indels) can cause nonsense mutations and truncation of
protein products or the introduction of a stop codon to produce gene
knockouts.


Figure 2. Gene editing workflow using the Edit-R CRISPR Cas9 system
Figure 2. Gene editing workflow using the Edit-R CRISPR Cas9 system

Edit-R Cas9 Nuclease Expression Plasmids

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. There are
several Edit-R Cas9 Nuclease Expression plasmid options from which to
choose, based on experimental preferences and cell types:


  1. SMARTCas9-mKate2 Expression plasmids:
    Cas9 nuclease and the mKate2 fluorescent reporter are both expressed
    under the control of a single RNA pol II promoter, thus making this
    plasmid useful for downstream cell enrichment by FACS. Six different
    RNA pol II promoter options are offered so that one which is highly
    active in the cells of interest can be selected.
  2. SMARTCas9-PuroR Expression plasmids:
    Cas9 nuclease and the Puromycin-resistance marker are both expressed
    under the control of a single RNA pol II promoter, thus making this
    plasmid useful for downstream cell enrichment by antibiotic (Puromycin)
    selection. Six different RNA pol II promoter options are offered so that
    one which is highly active in the cells of interest can be selected.
  3. Edit-R Cas9 Nuclease (hCMV-BlastR Expression plasmid: 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.
Learn more about all of the Edit-R Cas9 Nuclease Expression plasmid options »


Edit-R trans-activating CRISPR RNA (tracrRNA)

The Edit-R tracrRNA is a chemically synthesized and HPLC-purified long RNA molecule based on the published S. pyogenes tracrRNA sequence (Jinek, 2012). The Edit-R tracrRNA has been tested for efficient editing in multiple mammalian cell types.


Edit-R CRISPR RNA (crRNA)

The active Edit-R crRNA is a chemically synthesized RNA, comprised of 20 nucleotides identical to the
genomic DNA target site, or protospacer, followed by the required S. pyogenes
repeat
sequence that interacts with the tracrRNA. The chosen
20-base target sequence in the gene must be immediately upstream of a
protospacer-adjacent motif (PAM) in the genomic DNA. The predominant S. pyogenes PAM nucleotide sequence is NGG.


Edit-R™ CRISPR-Cas9 Gene Engineering Platform

Edit-R CRISPR-Cas9 Gene Engineering Platform

This video gives an overview of CRISPR-Cas9 gene
editing, and details how the Edit-R™ CRISPR-Cas9 Gene Engineering
Platform simplifies the workflow with the use of synthetic RNA.


View video (3:19)


Introduction to CRISPR

The ability to precisely and permanently alter endogenous
gene expression through targeted genome editing is a highly effective
reverse genetics tool. Recently, genome engineering has advanced
tremendously with the characterization of bacterial and archael CRISPR
(clustered regularly interspaced short palindromic repeats) systems and
their adapted usage in mammalian cells. (Figure 1)


Gene Editing - CRISPR-Cas system of bacterial adaptive immunity
Figure 1. Features of the CRISPR-Cas adaptive immune system. Adapted from Bhaya et al., Annu. Rev. Genet. 2011. 45:273-97

CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats



Cas: CRISPR-associated proteins

  1. Acquisition via integration of foreign DNA-derived spacers into CRISPR locus
  2. Expression (transcription and processing) of guide CRISPR RNAs (crRNAs) consisting of single spacer-repeat unit
  3. Interference of incoming viral or plasmid activity through cleavage of matching sequence
Gene Editing - Figure 2 -Engineering a CRISPR-Cas9 Platform for Mammalian Genome Engineering
Figure 2. A Type II
CRISPR-Cas9 system generally consists of the Cas9 nuclease complex
programmed by tracrRNA and crRNA. As such, it is easily adapted for
genome engineering in mammalian cells.
CRISPR systems have been described in the literature as
innate immune defense systems analagous to eukaryotic RNA interference
(RNAi) pathways. Additionally, the Cas9 (CRISPR-associated 9) nuclease
has been defined as a dedicated effector enzyme that cleaves DNA when
guided by two required small RNA sequences: the CRISPR RNA (crRNA) which
binds the target DNA and guides cleavage, and the trans-activating RNA
(tracrRNA) which base-pairs with the crRNA and enables the Cas9-crRNA
complex to locate the targeted DNA. Recent publications demonstrate
this system can be engineered to target and cleave DNA in mammalian
cells, thereby permanently disrupting gene expression (Figure 2), making
this system a new and exciting molecular tool to interrogate gene
function.


Recommended Reading

Below is a selection of important journal articles in the CRISPR-Cas9 research field.


  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 REAL-Fast 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).