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

The CRISPR Journal, vol. 5, pp. 571–585

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- 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 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 .

Authors 9

1. Thomas Swartjes

   Wageningen University & Research

   Affiliation as printed

   Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands; Delft, The Netherlands

   Delft, The Netherlands

2. Peng Shang Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

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

   Delft, The Netherlands

3. Dennis T.M. van den Berg

   Wageningen University & Research

   Affiliation as printed

   Food Microbiology, Wageningen University and Research, Wageningen, The Netherlands; Delft, The Netherlands

   Delft, The Netherlands

4. Tim Künne

   Wageningen University & Research

   Affiliation as printed

   Food Microbiology, Wageningen University and Research, Wageningen, The Netherlands; Delft, The Netherlands

   Delft, The Netherlands

5. Niels Geijsen Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

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

   Delft, The Netherlands

6. Stan J. J. Brouns

   Delft University of Technology

   Affiliation as printed

   Department of Bionanoscience, Delft University of Technology, Delft, The Netherlands; and Delft, The Netherlands

   Kavli Institute of Nanoscience, Delft, The Netherlands

   Delft, The Netherlands

7. John van der OOST

   Wageningen University & Research

   Affiliation as printed

   Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands; Delft, The Netherlands

   Delft, The Netherlands

8. Raymond H.J. Staals corresponding

   Wageningen University & Research

   Affiliation as printed

   Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands; Delft, The Netherlands

   Delft, The Netherlands

9. Richard A. Notebaart corresponding

   Wageningen University & Research

   Affiliation as printed

   Food Microbiology, Wageningen University and Research, Wageningen, The Netherlands; Delft, The Netherlands

   Delft, The Netherlands