Complex dependence of CRISPR-Cas9 binding strength on guide RNA spacer lengths

Aset Khakimzhan, David Garenne, Benjamin Tickman, Jason Fontana, James Carothers, Vincent Noireaux

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

It is established that for CRISPR-Cas9 applications guide RNAs with 17-20 bp long spacer sequences are optimal for accurate target binding and cleavage. In this work we perform cell-free CRISPRa (CRISPR activation) and CRISPRi (CRISPR inhibition) experiments to demonstrate the existence of a complex dependence of CRISPR-Cas9 binding as a function of the spacer length and complementarity. Our results show that significantly truncated or mismatched spacer sequences can form stronger guide-target bonds than the conventional 17-20 bp long spacers. To explain this phenomenon, we take into consideration previous structural and single-molecule CRISPR-Cas9 experiments and develop a novel thermodynamic model of CRISPR-Cas9 target recognition.

Original languageEnglish (US)
Article number056003
JournalPhysical Biology
Volume18
Issue number5
DOIs
StatePublished - Jul 2 2021

Bibliographical note

Publisher Copyright:
© 2021 IOP Publishing Ltd.

Keywords

  • cell-free transcription translation
  • CRISPR-Cas9
  • CRISPRa
  • CRISPRi
  • synthetic biology
  • thermodynamics

PubMed: MeSH publication types

  • Journal Article
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, Non-U.S. Gov't

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