Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

Swartjes, T.; Shang, P.; van den Berg, D.; Kunne, T. A.; Geijsen, N.; Brouns, S. J. J.; van der Oost, J.; Staals, R.; Notebaart, R. A.

doi:10.1101/2022.01.15.475214

bioRxiv · 10.1101/2022.01.15.475214

Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

Swartjes, T.·Shang, P.·van den Berg, D.·Kunne, T. A.·Geijsen, N.·Brouns, S. J. J.·van der Oost, J.·Staals, R.·Notebaart, R. A.

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Abstract

CRISPR-Cas has revolutionized genome editing and has a great potential for 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. Here we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides down-regulate Cas9 activity in human cells, reducing both on and 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 PAM-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

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Swartjes, T., Shang, P., van den Berg, D., Kunne, T. A., Geijsen, N., Brouns, S. J. J., van der Oost, J., Staals, R., Notebaart, R. A.. 2022-01-15. Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides. https://doi.org/10.1101/2022.01.15.475214

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