GenWand™ dsDNA demonstrated 80% higher KI efficiency compare to in-house PCR dsDNA (38% vs 21%)
GFP KI at RAB11A Locus in HEK293 cells using electroporation with 5ug of PCR dsDNA and GenWand™ dsDNA templates.
Double-stranded DNA (dsDNA) has been used for CRISPR homology directed repair (HDR) template for creating gene knock-in with high editing efficiency. GenWand™ dsDNA is developed with covalently closed ends to mitigate no-homologous end joining risk and increase knock-in accuracy. GenScript now offers up to g level closed-end dsDNA ideal for screening, process optimization and scale up.
dsDNA
Starting at 50µg/item , Deliver in as fast as 3 weeks .
| Length (bp) | Research Grade |
|---|---|
| 2000~5000 | Starting from $1,800/item |
| 5001~10000 | Starting from $2,200/item |
| Test Specifications | Detection Method | Release Criteria | Research Grade |
|---|---|---|---|
| Purity | Agarose gel electrophoresis | Single band |
✔
|
| Sequence accuracy | Sanger sequencing | 100% sequence alignment |
✔
|
| Optical density | Spectrophotometer at 260 nm/230 nm | ≥ 2.0 |
✔
|
CRISPR based gene insertion, replacement, or correction
Mechanism of CRISPR HDR based gene editing
CRISPR/Cas9 technology is commonly used to create precise double stranded breaks (DSBs) at target DNA sites. The guide RNA (gRNA) recognizes the protospacer adjacent motif (PAM) sequence on the target DNA after forming complex with Cas9, then Cas9 exerts its endonuclease function to cause DSBs. This triggers two mechanisms for repair: one is non-homologous end-joining (NHEJ), which introduces mutations in the DSB site. The other mechanism is homology directed repair (HDR) which enables the donor DNA to be inserted at the break site and create gene knock-ins.
GenWand™ dsDNA demonstrated 80% higher KI efficiency compare to in-house PCR dsDNA (38% vs 21%)
GFP KI at RAB11A Locus in HEK293 cells using electroporation with 5ug of PCR dsDNA and GenWand™ dsDNA templates.
CRISPR Knock-in Protocol for HEK 293T/293 cells with Thermo Fisher Neon®
Transfection System
CRISPR Knock-in Protocol for Jurkat cells with Thermo Fisher Neon® Transfection
System
CRISPR Knock-in Protocol for stimulated Human T Cells with Lonza 4D Nucleofector™
X Unit
CRISPR Knock-in Protocol for stimulated Human T Cells with Maxcyte® Electroporation System
Design and Preparation Guidelines for CRISPR KI HDR Templates
