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

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

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 .

Authors 9

1. Thomas Swartjes

   Wageningen University & Research

   Affiliation as printed

   Laboratory of Microbiology, Wageningen University & Research, Wageningen, The Netherlands

2. Peng Shang Aachen Department of Anatomy and Embryology

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Department of Anatomy and Embryology, Leiden University Medical Centre, Leiden, The Netherlands

3. Dennis J. van den Berg

   Wageningen University & Research

   Affiliation as printed

   Food Microbiology, Wageningen University & Research, Wageningen, The Netherlands

4. Tim Künne

   Wageningen University & Research · Delft University of Technology

   Affiliation as printed

   Department of Bionanoscience, Delft University of Technology, The Netherlands

   Laboratory of Microbiology, Wageningen University & Research, Wageningen, The Netherlands

5. Niels Geijsen Aachen Department of Anatomy and Embryology

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Department of Anatomy and Embryology, Leiden University Medical Centre, Leiden, The Netherlands

6. Stan J. J. Brouns

   Delft University of Technology

   Affiliation as printed

   Department of Bionanoscience, Delft University of Technology, The Netherlands

   Kavli Institute of Nanoscience, Delft, The Netherlands

7. John van der OOST

   Wageningen University & Research

   Affiliation as printed

   Laboratory of Microbiology, Wageningen University & Research, Wageningen, The Netherlands

8. Raymond H.J. Staals corresponding

   Wageningen University & Research

   Affiliation as printed

   Laboratory of Microbiology, Wageningen University & Research, Wageningen, The Netherlands

9. Richard A. Notebaart corresponding

   Wageningen University & Research

   Affiliation as printed

   Food Microbiology, Wageningen University & Research, Wageningen, The Netherlands