Modulating CRISPR-Cas Genome Editing Using Guide-Complementary DNA Oligonucleotides

Thomas Swartjes, Peng Shang, Dennis T.M. Van Den Berg, Tim Künne, Niels Geijsen, Stan J.J. Brouns, John van der Oost, Raymond H.J. Staals*, Richard A. Notebaart

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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.

Original languageEnglish
Pages (from-to)571-585
JournalCRISPR Journal
Volume5
Issue number4
DOIs
Publication statusPublished - 12 Aug 2022

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