Background information
Cas9 originates from the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) adaptive immunity system in
Streptococcus pyogenes . The original Cas9 Nuclease has two catalytic domains, the RuvC-like nuclease domain that cleaves the non-complementary strand and the HNH nuclease domain that cleaves the complementary strand of DNA. Cas9 Nickase carries a D10A point mutation that eliminates the nuclease function of the RuvC-like domain, so that only the strand of the DNA that is complementary to the guide RNA sequence is cleaved.
Cas9 Nickase is used to induce site-directed single strand breaks within the genomic target DNA. By introducing a sequence of interest linked to homologous sequences of the target region, single strand break repair enhances homologous recombination and genomic integration of the sequence of interest. In contrast to Cas9 Nuclease, non-homologous end joining (NHEJ) is not involved in the repair of single strand breaks introduced by Cas9 Nickase. Therefore, potential off-target single strand breaks will be reliably repaired by the base excision repair (BER) pathway without the risk of offside genomic modifications.
A pair of guide RNAs can be used to introduce two single strand breaks. Using two guide RNAs designed to recognize the opposite strands with less than 8 bp overlap between the guide RNA sequences and to give 5´-overhangs has been shown to efficiently induce indel formation by non-homologous end joining (NHEJ). Thereby, specificity of gene knock outs can be dramatically improved.
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.
