BioProject

Although CRISPR systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. More...

Although CRISPR systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. In some cases,, these bacterial nucleases have demonstrated off-target activity. when applied in mammalian cells in some instances. This creates a potential hazard for therapeutic applications and could confound results in biological research. Therefore, improving the precision of these nucleases is of broad interest. To complement approaches that increase specificity by modifying the enzyme, Here we show that engineering a hairpin secondary structure onto the spacer region of single guide RNAs (hp-sgRNAs) can increase specificity by several orders of magnitude when combined with various CRISPR effectors. The hairpin poses a steric and energetic barrier to R-loop formation that can only be overcome at sites with perfect complementarity to the sgRNA. We first demonstrate that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from S. pyogenes (SpCas9). We then show that hp-sgRNAs increase the specificity of gene editing using five different Cas9 or Cas12a variants. Our results demonstrate that RNA secondary structure is a fundamental parameter that can tune the activity of diverse CRISPR systems. Less...