CRISPRCLEAR™ Custom Transfection-Ready Kit contains next generation sequencing validated gRNA plasmid(s) designed to target a gene in your cell line of interest, donor DNAs and Cas9 expression plasmids. The Cas9 plasmids contain wild type Cas9. For researchers who prefer a more stringent off-target profile, we also provide Cas9 D10A or H840A nickase plasmids. Each kit is custom made to specifically target your gene-of-interest.
The gRNAs are functionally validated in N2A (mouse) or K562 (human) cells
To evaluate the cutting efficiency in the cells, next generation sequencing (NGS) was chosen to replace Surveyor assay. Genomic DNA were isolated from the cells transfected with the DNA construct contains gRNA and Cas9. PCR was performed using primers flanking the gRNA region. PCR products were then subject to next generation sequencing. The numbers of molecules with indel over total molecules read was used to calculate cutting efficiency. Compared to Surveyer assay, NGS give you more direct read out.
Custom Kits on demand:
- Point Mutation, Knockin, Knockout
- Cas9 D10A Nickase or Cas9 H840A Nickase
- Order by Gene ID#
- Human, Mouse, Rat
Please contact us to discuss your project.