I’ve identified the target sequence for a CRISPR Cas9 gene editing experiment. What components do I need?

To attain unmatched consistency between your CRISPR experimental replicates use TriLink’s CleanCap Cas9 mRNA and high quality sgRNA. sgRNA, also known as Single Guide RNA, a chimeric RNA composed of crRNA and tracrRNA, connected by a short RNA linker. The purpose of sgRNAs is to bind to Cas9 and direct the complex to a specific genomic location. Other technologies exists as a 2-part crRNA:tracrRNA guide RNAs.

TriLink’s sgRNA offers several advantages over 2-part guide RNAs. With a sgRNA there is no obligatory annealing step in vitro. This annealing step can be time consuming and inefficient. Especially when modified with 2’OMethyl and Phosphothioates, sgRNA is more stable to degradation by intracellular exonucleases (Porteus, Nature, June 2015). With enhanced stability, you will achieve better gene editing.

TriLink’s CleanCap Cas9 mRNA enables rapid gene expression and eliminates the risk of insertional mutagenesis that can be associated with DNA plasmid approach. The benefits of DNA-free, mRNA-based CRISPR over plasmid delivery include no danger of unintended DNA insertion, reduced toxicity, better on-target efficiency, and improved specificity. TriLink’s CleanCap Cas9 mRNA offers superior translational efficiency than conventional Cas9 mRNA. For maximal expression in cells or target organs, transfected mRNAs must avoid detection by pattern recognition receptors (PRRs) that evolved to sense improperly capped RNAs and double stranded RNA. PRR activation leads to cytokine production, translational arrest and cell toxicity or death. CleanCap mRNA is highly efficiently capped with a natural Cap1 structure and sequence engineered to improve activity.

Materials Required:

  • Synthetic guide RNA (sgRNA)
  • CleanCap Cas9 mRNA (modified L-7206 or wild type L-7606)
  • Tissue culture plates
  • Microcentrifuge Tubes
  • Cell Counter
  • Normal Growth Medium
  • Preferred Transfection Reagent (Lipofectamine, Nucleofection, TransIT, etc.)