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Increasing the specificity of CRISPR systems with engineered RNA secondary structures

 

D. Dewran Kocak, Eric A. Josephs, Vidit Bhandarkar, Shaunak S. Adkar, Jennifer B. Kwon & Charles A. Gersbach

Nature Biotechnology (2019)

 

Abstract:

CRISPR (clustered regularly interspaced short palindromic repeat) 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. 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. 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. We first demonstrate that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from Streptococcus 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.

Available at: https://www.nature.com/articles/s41587-019-0095-1#rightslink

DOI: https://doi.org/10.1038/s41587-019-0095-1

 

 

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