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 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, Pınar Akçakaya, Grzegorz Sienski, Marcello Maresca
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.