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Increasing the specificity of CRISPR systems with engineered RNA secondary structures.

Publication ,  Journal Article
Kocak, DD; Josephs, EA; Bhandarkar, V; Adkar, SS; Kwon, JB; Gersbach, CA
Published in: Nature biotechnology
June 2019

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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Published In

Nature biotechnology

DOI

EISSN

1546-1696

ISSN

1087-0156

Publication Date

June 2019

Volume

37

Issue

6

Start / End Page

657 / 666

Related Subject Headings

  • Streptococcus pyogenes
  • RNA, Guide, CRISPR-Cas Systems
  • RNA
  • Nucleic Acid Conformation
  • Gene Editing
  • CRISPR-Cas Systems
  • Biotechnology
 

Citation

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Kocak, D. D., Josephs, E. A., Bhandarkar, V., Adkar, S. S., Kwon, J. B., & Gersbach, C. A. (2019). Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nature Biotechnology, 37(6), 657–666. https://doi.org/10.1038/s41587-019-0095-1
Kocak, D Dewran, Eric A. Josephs, Vidit Bhandarkar, Shaunak S. Adkar, Jennifer B. Kwon, and Charles A. Gersbach. “Increasing the specificity of CRISPR systems with engineered RNA secondary structures.Nature Biotechnology 37, no. 6 (June 2019): 657–66. https://doi.org/10.1038/s41587-019-0095-1.
Kocak DD, Josephs EA, Bhandarkar V, Adkar SS, Kwon JB, Gersbach CA. Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nature biotechnology. 2019 Jun;37(6):657–66.
Kocak, D. Dewran, et al. “Increasing the specificity of CRISPR systems with engineered RNA secondary structures.Nature Biotechnology, vol. 37, no. 6, June 2019, pp. 657–66. Epmc, doi:10.1038/s41587-019-0095-1.
Kocak DD, Josephs EA, Bhandarkar V, Adkar SS, Kwon JB, Gersbach CA. Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nature biotechnology. 2019 Jun;37(6):657–666.

Published In

Nature biotechnology

DOI

EISSN

1546-1696

ISSN

1087-0156

Publication Date

June 2019

Volume

37

Issue

6

Start / End Page

657 / 666

Related Subject Headings

  • Streptococcus pyogenes
  • RNA, Guide, CRISPR-Cas Systems
  • RNA
  • Nucleic Acid Conformation
  • Gene Editing
  • CRISPR-Cas Systems
  • Biotechnology