gRNA Design and Off-Target Minimization for Genome Editing With CRISPR/Cas9

CRISPR/Cas9 is a relatively new genome editing technique which utilizes a short guide RNA (gRNA) sequence to direct an endonuclease into inducing a double stranded DNA break at a user selected located within a host genome. Innate cellular repair mechanisms are then activated, often resulting in the loss of gene function or incorporation of exogenetic material. Successful genome manipulation requires an abundance of accuracy and precision. However, gRNAs with inefficient site recognition can result in disastrous off-target mutations and unanticipated downstream effects. Since gRNAs have the potential to target a myriad of sites within a single gene, determining the most efficient gRNA with minimum off-target effects is paramount. Evaluation of gRNA efficiency can be determined through the use of predication models, gRNA sequence content, target location within host genome, and PAM site characteristics. Often determining the best gRNA candidates requires a balance between target efficiency and genomic location. This can be a daunting task, especially when working with a large number of gRNA candidates. Fortunately, public online resources, such as ChopChop and Benchling, have simplified the process of gRNA design by applying efficiency parameters in a user-friendly interface. My goal is to educate researchers on the fundamentals of gRNA design and walk them through examples of practical CRISPR/Cas9 applications using the available online resources.