CRISPR Point Mutation, Knockin, Knockout Kit

CRISPRCLEAR image

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 (Reference)
  • Order by Gene ID#
  • HumanMouseRat 

The gRNAs are functionally validated in N2A (mouse) or K562 (human) cells

To evaluate the cutting efficiency in the cells, next generation sequencing 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. 

 

An example of knock-out of a target gene in cancer cells with CRISPRCLEAR™ knockout kit 

HeLa cells were transfected with gRNA construct and Cas9 plasmid from ASK-7010 Cas9 CRISPRCLEARTM KO kit. Single cell clones were isolated. PCR products were sequenced. A single cell clone was identified with 5 bp deletion in a coding region at the gRNA site.

A). Chromatographs of wild type and mutant sequences. B). Alignment of the wild type and mutant sequences.

CRISPRCLEAR kit-KO data

 

An example of point mutation in a cell line with CRISPRCLEAR™  point mutation kit

Single cell clones were isolated. PCR products were sequenced. A single cell clone was identified with the right CTC>GCT correction. A. Chromatographs of mutant and corrected sequences. B. Alignment of the mutant and corrected sequences.

CRISPRCLEAR kit-PM data

An example of knock-in of reporter gene in human stem cells using CRISPRCLEAR™ knock-in kit

Human iPS cells generated by Applied Stemcell were transfected with H11 gRNA construct, mCherry donor construct and Cas9 plasmid from ASK-7030 Cas9 CRISPRCLEARTM Knock-in kit. Single cells were sorted and cultured. Single-cell clones were isolated. A. Microscopic picture of a colony of parental human iPS cell. B. Microscopic picture of Site-Specific Knock-In colonies expressing florescent mCherry protein.  

CRISPRCLEAR kit-KI data-1 CRISPRCLEAR kit-KI data-2

References
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