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

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 ben...

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Detalles Bibliográficos
Autores principales: Swartjes, Thomas, Shang, Peng, van den Berg, Dennis T.M., Künne, Tim, Geijsen, Niels, Brouns, Stan J.J., van der Oost, John, Staals, Raymond H.J., Notebaart, Richard A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Mary Ann Liebert, Inc., publishers 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9419950/
https://www.ncbi.nlm.nih.gov/pubmed/35856642
http://dx.doi.org/10.1089/crispr.2022.0011
Descripción
Sumario: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.