Single-guide RNAs: rationale and design

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene editing is a powerful tool with immense potential in the development of novel treatment strategies for a range of diseases. Efficiency of gene editing and site targeting using the Cas9 endonuclease is dependent on the single-guide RNA (sgRNA) sequence. The sgRNA is a synthetic complex of the native CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) necessary for site-specific CRISPR/Cas9 double-stranded breaks. One of the greatest challenges using CRISPR/Cas9 technology has been minimizing off-target activity, especially at sites with sequence homology to the area of interest. The crRNA sequence is able to tolerate mismatches with the target DNA, leading to off-target activity. Intrinsic characteristics to be considered when designing sgRNAs include the number and distribution of potential off-target mismatch pairing sites, propensity to form secondary structures, the nucleotides at specific positions, the GC content, and the characteristics of potential target and off-target sites. Thus optimizing sgRNA design, which mediates guiding of Cas9 to the target site, is crucial for efficient CRISPR/Cas9 activity.