Modulating CRISPR-Cas Genome Editing Using Guide-Complementary DNA Oligonucleotides
Thomas Swartjes (Wageningen University & Research)
Peng Shang (Leiden University Medical Center)
Dennis T.M. Van Den Berg (Wageningen University & Research)
Tim Künne (Wageningen University & Research)
Niels Geijsen (Leiden University Medical Center)
Stan J.J. Brouns (Kavli institute of nanoscience Delft, TU Delft - BN/Stan Brouns Lab)
John Van Der Oost (Wageningen University & Research)
Raymond H.J. Staals (Wageningen University & Research)
Richard A. Notebaart (Wageningen University & Research)
More Info
expand_more
Other than for strictly personal use, it is not permitted to download, forward or distribute the text or part of it, without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license such as Creative Commons.
Abstract
Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) has revolutionized genome editing and has great potential for many applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. In this study, we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides inhibit Cas9 activity in human cells, reducing both on- A nd off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: The length of the complementary region and the presence of a protospacer adjacent motif-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.
help_outline