Journal
NATURE BIOTECHNOLOGY
Volume 37, Issue 6, Pages 657-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41587-019-0095-1
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Funding
- Allen Distinguished Investigator Award from the Paul G. Allen Frontiers Group
- US National Institutes of Health (NIH) Director's New Innovator Award [DP2OD008586]
- NIH [R01DA036865, R01AR069085, P30AR066527]
- National Science Foundation [DMR-1709527, EFMA-1830957]
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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.
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