2. Academic Validation
  3. A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo

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

  • Nat Commun. 2023 Sep 6;14(1):5474. doi: 10.1038/s41467-023-41240-7.
Burcu Bestas 1,  Sandra Wimberger # 1 2,  Dmitrii Degtev # 1,  Alexandra Madsen 1,  Antje K Rottner 1,  Fredrik Karlsson 3,  Sergey Naumenko 4,  Megan Callahan 5,  Julia Liz Touza 6,  Margherita Francescatto 3,  Carl Ivar Möller 1,  Lukas Badertscher 1,  Songyuan Li 1,  Silvia Cerboni 7,  Niklas Selfjord 1,  Elke Ericson 1,  Euan Gordon 8,  Mike Firth 3,  Krzysztof Chylinski 9,  Amir Taheri-Ghahfarokhi 1,  Mohammad Bohlooly-Y 6,  Mike Snowden 10,  Menelaos Pangalos 11,  Barrett Nuttall 5,  Pinar Akcakaya 1,  Grzegorz Sienski 12,  Marcello Maresca 13
Affiliations

Affiliations

  • 1 Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D Unit, AstraZeneca, Gothenburg, Sweden.
  • 2 Department of Chemistry & Molecular Biology, University of Gothenburg, Gothenburg, Sweden.
  • 3 Data Sciences and Quantitative Biology, Discovery Sciences, BioPharmaceuticals R&D Unit, AstraZeneca, Cambridge, UK.
  • 4 Department of Biostatistics, Harvard Chan School of Public Health, Boston, MA, USA.
  • 5 Translational Genomics, Translational Medicine, R&D Oncology, AstraZeneca, Waltham, MA, USA.
  • 6 Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D Unit, AstraZeneca, Gothenburg, Sweden.
  • 7 Translational Science and Experimental Medicine, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 8 Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D Unit, AstraZeneca, Gothenburg, Sweden.
  • 9 Vienna Biocenter Core Facilities, Vienna Biocenter (VBC), Vienna, Austria.
  • 10 Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK.
  • 11 BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK.
  • 12 Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D Unit, AstraZeneca, Gothenburg, Sweden. grzegorz.sienski@astrazeneca.com.
  • 13 Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D Unit, AstraZeneca, Gothenburg, Sweden. marcello.maresca@astrazeneca.com.
  • # Contributed equally.
PMID: 37673883 DOI: 10.1038/s41467-023-41240-7
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.

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