Genome editing

The use of the CRISPR/Cas9 system in mammalian cells has recently emerged as a very convenient way to modify the cell genome at a specific locus. It involves transient transfection into mammalian cells of either (a) one or several plasmids coding for Cas9, the specific gRNA and eventually the sequence to be inserted, or (b) a mix of one or two plasmids and an RNA molecule (the gRNA).


Genome editing using CRISPR/Cas9 system

The association of Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) nucleases is an innovative technology to generate gene knock-outs or to introduce defined sequence modifications or deletions in the genome. The CRISPR/Cas9 system is a prokaryotic immune mechanism that confers resistance to foreign genetic elements and consists of a DNA nuclease called Cas9 and a non-coding guide RNA (gRNA). The gRNA guides the Cas9 nuclease to a specific complementary genomic locus where it induces a double strand break.

When using CRISPR/Cas9 system, transfection is a key and often limiting step to ensure a successful genome editing.

Our jetPRIME® reagent is especially well-suited for genome editing in vitro as it is efficient, requires low DNA amount, is suitable for multiple plasmid co-transfection and very gentle on cells. Indeed, several groups already successfully used jetPRIME®-mediated transfection of the CRISPR/Cas9 system (Davidson et al., (2014) Genes Dev 28(4):342-56; Moore et al., (2014) Nucleic Acids Res 43, 1297-303; Shi, J., et al. (2014), Nature 514, 187-9).

The optimal transfection conditions depend on the cell line to be transfected. Specific conditions for many cell types can be found in our Cell Transfection Database or you may contact our delivery experts at support@polyplus-transfection.com for a tailored protocol for your experiments.

For genome editing in vivo we recommend using in vivo-jetPEI® reagent as it leads to excellent delivery of nucleic acids to various organs in vivo using different delivery routes.

Citations

Li, Y. (2016) Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions. PLoS One 11 e0144970

Shi, J. & al.  (2015). Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell deathNature 526 660-5

D’Osualdo, A. (2015). Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress. PLoS ONE 10 e0130635

Moore, R., (2015) CRISPR-based self-cleaving mechanism for controllable gene delivery in human cells. Nucleic Acids Res 43 1297-303


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