Custom sgRNA/Cas9

RGENs (RNA Guided Endonucleases)

RNA-Guided ENdonucleases (RGENs) are novel, programmable genome engineering tools that are adapted from CRISPR/Cas system. Cas9 forms a sequence-specific endonuclease when complexed with two RNAs, one of which guides target selection (crRNA) and the other is a constant component (tracrRNA).
RGENs recognize a target specific sequence that is 23bp in length, ending with two guanines (GG). This minimal requirement for target site selection means that RGENs can be designed to cleave any region of the genome (every 8bp in theory).

CRISPR/Cas9 gRNA Cas9 protein

RGENs have recently been shown to support efficient genome engineering in human cells, zebrafish and bacteria. In addition, the use of RGENs allows multiplexing of genome engineering processes (e.g., knockout of multiple genes in a cell at one time).
RGEN also shows impressive sequence specific activity. The activity and specificity of RGEN is comparable to TALEN.

gRNA Cas9 protein

HEK293 cells are transfected with increasing amount of plasmid expressing sgRNA in the presence or absence of Cas9 plasmid. In/del caused by CRISPR/Cas9 complex was assessed by T7E1 assay.

We provide custom CRISPR/Cas9 (RGEN) synthesis service with or without validation.

IVT sgRNA Synthesis Service (aRGEN)

  • Includes design, off-target search, and synthesis
  • Provides 50 ug sgRNA, lyophilized form
  • Injection/transfection ready
  • Quality/quantity confirmed in agarose gel
  • Time line: 2 weeks
  • sgRNA Vector Synthesis Service (dRGEN)

  • Includes design, off-target search, and synthesis
  • Provides an expression plasmid of sgRNA and Cas9
  • Time line: 2 weeks
  • Cas9 vectors available with coexpression of GFP/HygroR and RFP/PuroR cassette under CMV or EF1a promoter for selection
  • sgRNA Validation Service

  • Customer provides gene name, species, and project goal (KO, KI etc)
  • Includes design, off-target search, and synthesis of 4~8 gRNA vectors
  • Transfect CRISPR/Cas9 into cells and perform mismatch sensitive nuclease assay to find the most efficient nuclease
  • sgRNA is provided in an expression plasmid (dRGEN) or RNA format (aRGEN)
  • PCR primers, PCR condition, and mismatch sensitive nuclease assay for KO screening is also provided
  • Time line: 3~6 weeks
  • Cas9 vectors

  • CV01 & CV11: Cas9 (CMV or EF1a promoter)
  • CV02 & CV12: Cas9-HygR-GFP (CMV or EF1a promoter)
  • CV03 & CV13: Cas9-RFP-PuroR (CMV or EF1a promoter)
  • Guide RNA (gRNA) Design

    In general, find gRNA sequence with no off-targets for 1 bp and 2 bp mismatches, and contains 45~70% GC. If you cannot avoid off-targets, mismatches located near PAM are preferred over the ones at 5' side of gRNA. Please note that non-canonical PAM (-NAG, -NGA) can also be cleaved.
    We have developed our gRNA Tool that outputs off-target sequences, GC content and other features. You can also use publically available tools to search with various options.
    Cas-Offinder (to see the off-target list)

    eGFP Control sgRNA

  • Validated for cutting eGFP both in vitro and in vivo
  • Injection ready form, lyophilized powder
  • Also available with eGFP plasmid and Cas9 protein as positive control reagents for in vitro cleavage assay
  • Available sizes: 20 ug or 100 ug (20 ug x 5 tubes)
  • Catalog number: AR04
  • Surrogate Reporter

  • To validate the activity of various nucleases (CRISPR/Cas9, TALEN, and ZFN) in cells
  • Also to enrich cells with gene editing events for clone screening
  • Use FACS, MACS or drug selection
  • Choice of empty plasmid or custom designed for the nuclease target sequence
  • Please refer to "Reporters for Nucleases" for more details
  • Time line: 2 weeks for custom surrogate reporter vectors
  • Cat No Description Price
    DR01 Custom sgRNA/Cas9 expression plasmids Inquire
    DR02 Activity validated sgRNA/Cas9 vector by mismatch sensitive nuclease assay in cells Inquire
    AR01 Custom IVT sgRNA, injection ready (50 ug) Inquire
    AR02 1~3 in vitro validated sgRNA, injection ready (50ug each) Inquire
    AR04 eGFP control sgRNA (20 ug or 100 ug) Inquire
    Surrogate reporter system
    RV01 GFP surrogate reporter system / 5 ug $350
    RV02 MACS/GFP surrogate reporter system / 5 ug $350
    RV03 HygR/GFP surrogate reporter system / 5 ug $350
    Cas9 vectors
    CV01 Cas9 co-transfection vector (CMV promoter) / 5 ug $350
    CV02 Cas9/HygR/GFP vector (CMV promoter) / 5 ug $350
    CV03 Cas9/RFP/PuroR vector (CMV promoter) / 5 ug $350
    CV11 Cas9 co-transfection vector (EF1a promoter) / 5 ug $350
    CV12 Cas9/HygR/GFP vector (EF1a promoter) / 5 ug $350
    CV13 Cas9/RFP/PuroR vector (EF1a promoter) / 5 ug $350

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