HighYield T7 sgRNA Synthesis Kit (SpCas9)

HighYield T7 sgRNA Synthesis Kit (SpCas9) is designed for cloning-free synthesis of SpCas9-specific single-guided RNAs (sgRNAs) via in vitro transcription. SgRNAs direct sequence-specific DNA recognition once complexed with Streptococcus pyogenes Cas9 (SpCas9) . The resulting sgRNA/SpCas9 ribonucleoprotein (RNP) complex can thus be used for site-specific cleavage, nicking or binding of dsDNA both in vitro and in living cells depending on the choice of SpCas9 variant (e.g. wildtype, nickase (D10A), nuclease deficient (D10A/H840A) ) . Cleavage, nicking or binding of SpCas9 variant occurs upstream of the SpCas9-specific DNA recognition sequence 5'-NGG-3' (protospacer adjacent motif (PAM) sequence, N = any nucleotide base) . Cloning-free synthesis of sgRNA-encoding DNA template for T7 RNA Polymerase-mediated in vitro transcription is easily performed via PCR assembly with provided SpCas9 scaffold and T7 promotor containing PCR primer. Only a target-specific oligonucleotide (approx. 60 nt) needs to be provided. Amplification is performed with Ultra DNA Polymerase (also known as Phusion High-Fidelity Polymerase) to ensure the highest sequence accuracy as well as blunt-end formation. The crude PCR mix can directly be used as template for in vitro transcription. HighYield T7 sgRNA Synthesis Kit (SpCas9) contains sufficient reagents for 50 PCR assembly and in vitro transcription reactions. Other (s) gRNA-encoding T7 DNA templates (e.g with a different scaffold or for different Cas endonucleases) can efficiently be in vitro transcribed with the HighYield T7 RNA Synthesis Kit (#orb611235) .