Watch our archived webinar on genome editing in hard-to-transfect cells:

Duration: 61 minutes

The CRISPR (clustered regularly interspaced palindromic repeats) technology is based on a bacterial defense pathway against viral invasion. It has been adapted for genome editing applications in eukaryotic cells and is an emerging technology  because it allows for some more flexibility compared to zinc finger nucleases (ZFNs) or transcriptional activator-like effector nucleases (TALENs).

 

 

 

In contrast to ZFN and TALEN, CRISPR-based genome editing is relying on two separate components working together:

  • The DNA targeting part is the so called guideRNA (gRNA) that can be designed as such that it is complimentary to an 18-21 bp stretch of the genomic DNA.
  • Once paired with the genomic DNA strand, it recruits Cas9 nuclease (CRISPR-associated nuclease 9) to the genomic DNA which introduces a double-strand break at a defined site.

Since the DNA-specific part is an RNA molecule it is much easier to design and produce than a ZF- or TALE-nuclease fusion protein. In addition, by transferring Cas9 nuclease together with several gRNAs targeting different sites multiple genome editing is possible.

    

CRISPR-based Genome Editing Requires Co-transfection

For CRISPR-based genome editing, various transfection scenarios are possible:

 

  • Transfer of one plasmid carrying both the gRNA and Cas9 nuclease
  • Co-transfection of two separate plasmids (one for the gRNA and one for Cas9)
  • Co-transfection of a plasmid carrying Cas9 and PCR cassette expressing the gRNA (Ran FA et al. 2013)
  • An additional donor/repair plasmid or a single strand oligonucleotide (ssODN) has to be co-transfected when aiming for insertion or replacement

 

 

 

 

 

 

Our Nucleofector™ Technology has been shown to work as a reliable transfection method for CRISPR-based genome editing tools

  • High transfection efficiencies for a broad range of cell types, including iPSCs
  • Efficient co-transfection of various substrates
  • Same conditions for transfecting plasmids, DNA, mRNA or PCR cassettes, ssODN

The Nature Protocols publication from Ran et al.3 provides a very comprehensive guideline on using the 4D-Nucleofector™ System in combination with CRISPR technology . For further technical assistance on using the Nucleofector™ Technology for CRISPR-based genome editing, contact Lonza Scientific Support.



CRISPR References

1) Petit CS et al. (2013) J Cell Biol 202:1107-1122 (cells: HeLa)

2) Ran FA et al. (2013) Cell 154:1380–1389 (cells: various cell lines, e.g. HEK293FT)

3) Ran FA et al. (2013) Nat Prot 8(11):2281–2308 (cells: HEK293 and HUES62)