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

FAQ

1. What is the maximum size of knock-in fragment that can be used with the CRISPCLEAR™ Knock-in kit?

 The site-specific CRISPRCLEAR™ knock-in kit and the CRISPRCLEAR™ Safe-harbor locus knock-in kit can be used to insert up to 5-6 kb of DNA, including ~ 1000 bp length of homologous arms, into the cell line of choice.

 2. What details does a customer need to provide for a custom designed CRISPRCLEAR™ Kit?

The customer needs to provide the details regarding gene name, gene ID (relevant to the species), desired mutations, desired zygosity (heterozygous or homozygous), and preferably, the cell line that will be modified.

3. In which cell lines are the gRNAs validated?

The gRNAs for the CRISPRCLEAR™ Kits are validated in vitro using routinely used cell lines from the species whose gene is targeted. For human cells, we use K562 or HEK293 cells and for mouse cells, we use the N2A cell line to validate the gRNAs. For other species, we would use a routinely used cell line from the desired species.

4. Is a donor DNA required for the knockout kit?

A donor DNA is not required for gene knockout based on non-homologous end-joining mediated double stranded break (DSB) repair, to introduce the mutation.  This non-specific knockout will result in general loss of function of the gene by the introduction of indels causing frame-shift mutations. If, however a knockout requires deletion of specific sequence of nucleotides, a donor DNA will be provided with homologous arms to the desired region of interest, to enable homology directed repair (HDR).

5. How do you detect the efficacy of gRNA activity in in vitro evaluation assays?

We evaluate indel rates, and the subsequent gRNA activity, by next generation sequencing (NGS) or Surveyor Assay. In special cases, we will use the targeting cells or genomic DNA for evaluation.
 
6. Do you have kits for cell lines from other species such as non-human primates (NHPs), rabbits, etc.?
 
Our current catalog does not have CRISPRCLEAR™ Kits for species other than human and mouse. However, we have generated CRISPRCLEAR™ Kits for other species as a custom project. Please inquire with our technical solutions specialists for more details.
 
7. What components are provided with the CRISPRCLEAR™ Kit for Cell Lines?
 
The CRISPRCLEAR™ Kit for Cell Lines includes reagents sufficient for 3 rounds of transfection: (1) 10 µg validated gRNA constructs in plasmid format; (2) 15µg donor DNA plasmid or ssODN; (3) 10 µg Cas9-expressing plasmid.
 
8. How much does it cost for ordering extra donor or Cas9 plasmids?

The donor plasmid(s) are custom designed for each project and price varies with the features designed into the plasmid. Please inquire with our technical solutions specialists or your appointed project manager for more details regarding re-ordering donor and Cas9 plasmids.

References
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