Use Of FlashGel™ DNA Cassettes For Separations In Gene Editing Workflows.

The ability to perform genome editing has expanded tremendously in recent years and has been facilitated by the development of systems based on clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) that allow targeted cleavage of genomic loci. We have tested the use of FlashGel™ DNA Cassettes as tools to monitor the results of several electrophoretic separation steps that maybe involved in the CRISPR/Cas9 workflow: mismatch cleavage assays, single guide RNA (sgRNA) screening, and single stranded DNA (ssDNA) production. Mismatch cleavage assays are a quick and inexpensive tool to evaluate the efficiency of editing. FlashGel™ DNA Cassettes allowed rapid (five minutes) separation of cleavage products with high sensitivity. Separation performance was confirmed using control reagents from seven commercially available mismatch cleavage kits. Testing additionally showed that FlashGel™ DNA Cassettes worked well for rapid separation of cleavage products generated using control reagents from two sgRNA screening kits. ssDNA is becoming more widely used as a donor for knock-in CRISPR/Cas9 experiments involving homology directed repair. Testing in conjunction with control reagents from a commercially available ssDNA production system showed that FlashGel™ DNA Cassettes allowed monitoring of ssDNA generation and gave the added benefit of being able to distinguish ssDNA and dsDNA based upon observation of the color of the bands. The use of FlashGel™ DNA Cassettes in gene editing workflows offers increased separation speed and detection sensitivity relative to separations using conventional agarose gel formats.