Aldag, Pierre and Welzel, Fabian and Jakob, Leonhard and Schmidbauer, Andreas and Rutkauskas, Marius and Fettes, Fergus and Grohmann, Dina and Seidel, Ralf (2021) Probing the stability of the SpCas9-DNA complex after cleavage. NUCLEIC ACIDS RESEARCH, 49 (21). pp. 12411-12421. ISSN 0305-1048, 1362-4962
Full text not available from this repository. (Request a copy)Abstract
CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | R-LOOP FORMATION; DNA CLEAVAGE; CRISPR-CAS9; CAS9; TRANSCRIPTION; ENDONUCLEASE; SPECIFICITY; NUCLEASE; DYNAMICS; IMMUNITY; |
| Subjects: | 500 Science > 570 Life sciences 600 Technology > 615 Pharmacy |
| Divisions: | Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Mikrobiologie (Archaeenzentrum) > Prof. Dr. Dina Grohmann Chemistry and Pharmacy > Institute of Pharmacy > Pharmacology and Toxicology (Prof. Schlossmann, formerly Prof. Seifert) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 09 Aug 2022 07:53 |
| Last Modified: | 09 Aug 2022 07:53 |
| URI: | https://pred.uni-regensburg.de/id/eprint/46170 |
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