CRISPR/Cas9 Plasmid Systems

Components

• Guide-it CRISPR/Cas9 System (Green) (Cat. # 632601)
  
  • pGuide-it-ZsGreen1 Vector (Linear)
  • Guide-it Ligation Components
  • Stellar Competent Cells

• Guide-it CRISPR/Cas9 System (Red) (Cat. # 632602)
  • pGuide-it-tdTomato Vector (Linear)
  • Guide-it Ligation Components
  • Stellar Competent Cells

Genomic DNA modifications introduced using the pGuide-it-ZsGreen1 plasmid

Genomic DNA modifications introduced using the pGuide-it-ZsGreen1 plasmid. 1 x 105 cells (HEK 293, HeLa, U2OS, or HT1080 cell lines) were seeded in 12-well plates 16 hr prior to transfection. Cells were transfected with 2.5 µg of pGuide-it-ZsGreen1 plasmid using the Xfect Transfection Reagent; the plasmid expressed Cas9, ZsGreen1, and an sgRNA targeting the either CCR5, C4BPB, or EMX1. After 48 hr, fluorescence was analyzed by microscopy (A), and by FACS (B, Transfection efficiency). The efficiency of gene modification was also evaluated using the Guide-it Mutation Detection Kit, a PCR-based method for testing for the presence of indels (B). The target sequence was amplified directly from cells, the amplicon was melted and hybridized to form mismatched targets that were cleaved with Guide-it Resolvase. Cleavage products were resolved on an agarose gel and quantified by densitometry to estimate the frequency of indels (B, Indel).

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells. The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).