RNA Secondary Structure Increases the Specificity of Class 2 CRISPR Effectors

CRISPR systems have been widely adopted for both therapeutic applications and biological studies. In some cases, these bacterial nucleases have demonstrated off-target activity when applied in mammalian cells. This creates a potential hazard for therapeutic applications and could possibly confound results in biological research. Therefore, improving the specificity of these nucleases is of broad interest. Here we show that guide RNAs (gRNAs) with engineered secondary structure in the targeting region (hairpin-gRNAs) can have increased specificity by several orders of magnitude when combined with various CRISPR effectors. Focusing first on Cas9 from S. pyogenes (SpCas9), we show that rationally designed hp-gRNAs form in human cells and tune Cas9 binding. We then show that hp-gRNAs increase the specificity of gene editing using five different CRISPR endonucleases: (i) SpCas9, (ii) Cas9 from S. aureus (SaCas9), (iii) an SaCas9 variant with relaxed PAM specificity (SaCas9-KKH), (iv) Cpf1 from Acidaminococcus sp.BV3L6 (AsCpf1), and (v) Cpf1 from Lachnospiraceae bacterium ND2006 (LbCpf1). Our results suggest RNA secondary structure as a fundamental parameter that can tune the activity of class 2 CRISPR systems.