Assay Genie’s Express CRISPR sgRNA Synthesis Kit (S. pyogenes) is a neat handy tool for generating sgRNAs for spCas9 system. The 2X Express sgRNA Synthesis SuperMix contains all the components required for the in vitro transcription including an oligo template of the tracer sgRNA. To set up the reaction, simply combine the 2X Express sgRNA Synthesis SuperMix, Enzyme Mix, DNA Polymerase, the target-specific oligo, and water in a 20 μl reaction. The target-specific oligo first anneals with the sgRNA scaffold complementary oligo included in the 2X Express sgRNA Synthesis SuperMix, and DNA extension will be performed by the DNA polymerase. The newly extended dsDNA will then serve as the template for sgRNA transcription. High quality sgRNA can be obtained in as little as 30 minutes. The synthesized sgRNA can be used directly in downstream applications such as sgRNA in vitro screening. An optional DNase I treatment followed by spin column purification can be performed to remove DNA template, and leftover NTPs and dNTPs. This optional step could result in a more suitable sgRNA for downstream in vivo studies and applications.

Figure: All experiments should be performed in a nuclease-free environment. In addition, DNA sample preparation, reaction set-up and subsequent reaction(s) should be performed in separate areas to avoid cross-contamination. The use of “clean” pipettes designated for RNA work and aerosol-resistant barrier tips is recommended.