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

4 Scopus citations


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
Issue number5
StatePublished - Sep 2021

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© 2021 IOP Publishing Ltd.


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


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