Monitoring Guide RNA Synthesis for CRISPR/Cas9 Genome Editing Workflow Using the Agilent 2100 Bioanalyzer System

Bacterial clustered regularly interspaced short palindromic repeats (CRISPR) - associated protein 9 (Cas9) system has increased in popularity as a genome editing tool for targeted mutations, insertions, deletions and gene knock-out studies. CRISPR genome editing has also proved superior to Zinc-finger nucleases (ZFN) and transcription activator- like effector nucleases (TALENs) due to its simplicity and easy programmability. In CRISPR, a guide RNA (gRNA) is used to recognize and introduce a double standard break in a target DNA. The DNA repair mechanism triggered after the break is then exploited to introduce an insertion/deletion in the case of non-homologous end joining, or precise genetic modification if a homology-directed repair pathway is triggered.

A critical part of the CRISPR/Cas9 tool is the design and synthesis of the gRNA that comprises T7 promoter sequence, target sequence, and protospacer adjacent motifs. Monitoring the transcription of the gRNA is critical to the workflow to ensure successful gene editing. Here we present an automated electrophoresis approach for monitoring the synthesis, integrity, and functional activity of gRNAs created for a CRISPR/Cas9 workflow.