Background information
Cas9 originates from the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) adaptive immunity system of
Streptococcus pyogenes.
Cas9 nuclease can be used to introduce site-directed double strand breaks into the genomic DNA. These will subsequently be repaired through one of two repair pathways: non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ repair is error prone and will frequently result in nucleotide deletions or insertions (indel). If such insertions/deletions result in a frame shift or introduction of a premature stop codon, they will disrupt the open reading frame of the target gene. Therefore, this approach can be used to generate a gene knock out.
Alternatively, the HDR pathway can be used to introduce specific changes into the gene of interest. By providing a DNA repair template that contains the desired modification flanked by regions that are homologous to the genomic target sequence, HDR enables genomic integration of edited target sequence.
The position of the Cas9 cleavage site is determined by two factors: the target sequence recognized by the guide RNA and the presence of a Protospacer Adjacent Motif (PAM, in the case of Streptococcus pyogenes Cas9 the nucleotide sequence NGG). The Cas9/guide RNA complex will recognize the conjunction of both sequence elements and generate a double strand break 3 base pairs upstream of the PAM.
Detailed procedure
For satisfactory transfection results, use a transfection protocol optimized for your specific cell type. StemMACS™ eGFP mRNA or StemMACS™ Nuclear eGFP mRNA allow easy evaluation of transfection efficiency and are recommended as positive controls.
Quality description
StemMACS™ mRNAs are highly pure, in vitro-transcribed mRNAs that have been carefully optimized to ensure high level expression after transfection. Polyadenylation and enzymatic cap mimic a fully processed mature mRNA and ensure efficient translation. Incorporation of the modified nucleotides pseudouridine and 5-methylcytidine reduces the innate antiviral response to single-stranded mRNA. Robust expression of the encoded factor after transfection has been verified by immunofluorescence or flow cytometry.
StemMACS™ mRNAs are lyophilized from a 0.2 µM filtered solution for extended shelf-life.
