A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo

Burcu Bestas, Sandra Wimberger, Dmitrii Degtev, Alexandra Madsen, Antje K. Rottner, Fredrik Karlsson, Sergey Naumenko, Megan Callahan, Julia Liz Touza, Margherita Francescatto, Carl Ivar Möller, Lukas Badertscher, Songyuan Li, Silvia Cerboni, Niklas Selfjord, Elke Ericson, Euan Gordon, Mike Firth, Krzysztof Chylinski, Amir Taheri-Ghahfarokhi, Mohammad Bohlooly-Y, Mike Snowden, Menelaos Pangalos, Barrett Nuttall, Pinar Akcakaya, Grzegorz Sienski () and Marcello Maresca ()
Additional contact information
Burcu Bestas: AstraZeneca
Sandra Wimberger: AstraZeneca
Dmitrii Degtev: AstraZeneca
Alexandra Madsen: AstraZeneca
Antje K. Rottner: AstraZeneca
Fredrik Karlsson: AstraZeneca
Sergey Naumenko: Harvard Chan School of Public Health
Megan Callahan: AstraZeneca
Julia Liz Touza: AstraZeneca
Margherita Francescatto: AstraZeneca
Carl Ivar Möller: AstraZeneca
Lukas Badertscher: AstraZeneca
Songyuan Li: AstraZeneca
Silvia Cerboni: AstraZeneca
Niklas Selfjord: AstraZeneca
Elke Ericson: AstraZeneca
Euan Gordon: AstraZeneca
Mike Firth: AstraZeneca
Krzysztof Chylinski: Vienna Biocenter (VBC)
Amir Taheri-Ghahfarokhi: AstraZeneca
Mohammad Bohlooly-Y: AstraZeneca
Mike Snowden: AstraZeneca
Menelaos Pangalos: AstraZeneca
Barrett Nuttall: AstraZeneca
Pinar Akcakaya: AstraZeneca
Grzegorz Sienski: AstraZeneca
Marcello Maresca: AstraZeneca

Nature Communications, 2023, vol. 14, issue 1, 1-15

Abstract: Abstract Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.

Date: 2023
References: View references in EconPapers View complete reference list from CitEc
Citations: View citations in EconPapers (4)

Downloads: (external link)
https://www.nature.com/articles/s41467-023-41240-7 Abstract (text/html)

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-41240-7

Ordering information: This journal article can be ordered from
https://www.nature.com/ncomms/

DOI: 10.1038/s41467-023-41240-7

Access Statistics for this article

Nature Communications is currently edited by Nathalie Le Bot, Enda Bergin and Fiona Gillespie

More articles in Nature Communications from Nature
Bibliographic data for series maintained by Sonal Shukla () and Springer Nature Abstracting and Indexing ().