Rapid Generation of sgRNA

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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.

 

EnGen sgRNA Synthesis Kit Overview

Workflow
A. The target-specific oligo contains the T7 promoter sequence, ~20 nucleotides of target-specific sequence and a 14 nucleotide overlap sequence complementary to the S. pyogenes Cas9 Scaffold Oligo supplied in the reaction mix. Target-specific oligos (or EnGen sgRNA Control Oligo, S. pyogenes) are mixed with the EnGen 2X sgRNA Reaction Mix (NTPs, dNTPs, S. pyogenes Cas9 Scaffold Oligo) and the EnGen sgRNA Enzyme Mix (DNA and RNA polymerases) at room temperature. B. At 37°C the two oligos anneal at the 14 nucleotide overlap region of complementarity. C. The DNA polymerase extends both oligos from their 3´ ends creating a double-stranded DNA template. D. The RNA polymerase recognizes the double-stranded DNA of the T7 promoter and initiates transcription. The resulting sgRNA contains the target-specific/crRNA sequence as well as the tracrRNA. All steps occur in a single reaction during a 30 minute incubation at 37°C.

T7 transcription

GE_T7transcription_0514
In order for transcription to occur, a promoter sequence is required. Note that after transcription, the RNA retains the first three bases of the promoter, and is similar in sequence to the 5′ strand of DNA.