Publications
Physical biologyJun 2021 DOI:
10.1088/1478-3975/ac091e

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

Khakimzhan, Aset; Garenne, David; Tickman, Benjamin; Fontana, Jason; Carothers, James; Noireaux, Vincent
Product Used
Genes
Abstract
It is established that for CRISPR-Cas9 genetic guide RNAs with 17-20bp 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 18-20bp 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.
Product Used
Genes

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