TY - JOUR
T1 - Modulating CRISPR-Cas Genome Editing Using Guide-Complementary DNA Oligonucleotides
AU - Swartjes, Thomas
AU - Shang, Peng
AU - Van Den Berg, Dennis T.M.
AU - Künne, Tim
AU - Geijsen, Niels
AU - Brouns, Stan J.J.
AU - Van Der Oost, John
AU - Staals, Raymond H.J.
AU - Notebaart, Richard A.
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85138620960&partnerID=8YFLogxK
U2 - 10.1089/crispr.2022.0011
DO - 10.1089/crispr.2022.0011
M3 - Article
C2 - 35856642
AN - SCOPUS:85138620960
SN - 2573-1599
VL - 5
SP - 571
EP - 585
JO - CRISPR Journal
JF - CRISPR Journal
IS - 4
ER -