sgRNA Synthesis

In vitro transcription (IVT) is a powerful method to generate single guide RNA (sgRNA) that can be delivered with Cas protein, Cas mRNA, or Cas expression vectors for CRISPR/Cas experiments. Gene assembly, cloning and directed mutagenesis strategies enable quick construction or exchange of CRISPR/Cas targeting sequences in sgRNA vectors or for sgRNA libraries in CRISPR screening experiments to map phenotypes or disease-specific gene interactions, or identify novel therapeutic drug targets. 

Generate guide RNA for use with Spy Cas9 or Cas9 orthologs

Our extensive IVT portfolio includes several simple, cost-effective, and scalable solutions for synthesizing sgRNA compatible with either Streptococcus pyogenes Cas9 (Spy Cas9) or non-Spy Cas9 orthologs.

• EnGen^® sgRNA Synthesis Kit, S. pyogenes (NEB #E3322) is a simplified workflow that combines template synthesis and transcription with a single, user-supplied oligonucleotide. Use EnGen sgRNA Template Oligo Designer to configure target-specific oligos. Deliver sgRNA with any S. pyogenes Cas9 protein variant in our CRISPR/Cas Nuclease portfolio as a ribonucleoprotein complex (RNP) for high editing efficiency
• HiScribe^® T7 High Yield RNA Synthesis Kit (NEB #E2040) and HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB #E2050) use annealed oligos, PCR products or linearized plasmid DNA as templates to generate sgRNA or Cas9 mRNA.  
  
  

Construct Cas9-sgRNA vectors

A selection of our assembly and cloning solutions enable rapid encoding of target-specific sgRNAs for downstream in vivo or in vitro transcription.  

• The NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621) and Cloning Kit (NEB #E5520) are highly recommended for simplified construction of guide RNA vectors using single-stranded oligos. Short sequence sgRNA libraries can be constructed using pooled collections of ss oligos, each with specific overlaps for the sgRNA/Cas9 expression vector ends, to avoid chemical synthesis of double-stranded DNAs for individual variants.
• Q5^® Site-Directed Mutagenesis Kits (NEB #E0554 or NEB #E0552) quickly modify existing Cas9-sgRNA constructs up to 20 kb in length.