Increasing the specificity of CRISPR systems with engineered RNA secondary structures

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Eric Josephs, Assistant Professor (Creator)
The University of North Carolina at Greensboro (UNCG )
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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.

Additional Information

Nature Biotechnology 37, 657–666
Language: English
Date: 2019
CRISPR, single guide RNAs (sg-RNAs), gene editing, Streptococcus pyogenes

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