The EnGen sgRNA Synthesis Kit simplifies the generation of microgram quantities of custom sgRNAs in an hour or less by combining template synthesis and transcription. The single-tube reaction is easy to set up and requires a single ~55 nt ssDNA target-specific oligonucleotide, which is combined with the Reaction Mix and Enzyme Mix included in the kit. Target-specific DNA oligos are configured using NEB’s EnGen sgRNA Template Oligo Designer (accessible through NEBioCalculator®) and can be ordered from any DNA oligo supplier. sgRNAs are suitable for use in downstream applications, including CRISPR/Cas9-based genome editing and in vitro DNA cleavage. This single-reaction format offers ease-of-use and eliminates separate DNA amplification and template clean up steps.