Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases

Moritz J. Schmidt, Ashish Gupta, Christien Bednarski, Stefanie Gehrig-Giannini, Florian Richter, Christian Pitzler, Michael Gamalinda, Christina Galonska, Ryo Takeuchi, Kui Wang, Caroline Reiss, Kerstin Dehne, Michael J. Lukason, Akiko Noma, Cindy Park-Windhol, Mariacarmela Allocca, Albena Kantardzhieva, Shailendra Sane, Karolina Kosakowska, Brian Cafferty, Jan Tebbe, Sarah J. Spencer, Scott Munzer, Christopher J. Cheng, Abraham Scaria, Andrew M. Scharenberg, André Cohnen () and Wayne M. Coco ()
Additional contact information
Moritz J. Schmidt: Bayer AG
Ashish Gupta: Bayer AG
Christien Bednarski: Bayer AG
Stefanie Gehrig-Giannini: Bayer AG
Florian Richter: Bayer AG
Christian Pitzler: Bayer AG
Michael Gamalinda: Bayer AG
Christina Galonska: Bayer AG
Ryo Takeuchi: Casebia Therapeutics LLC
Kui Wang: Casebia Therapeutics LLC
Caroline Reiss: Casebia Therapeutics LLC
Kerstin Dehne: Bayer AG
Michael J. Lukason: CRISPR Therapeutics INC
Akiko Noma: Casebia Therapeutics LLC
Cindy Park-Windhol: Casebia Therapeutics LLC
Mariacarmela Allocca: Casebia Therapeutics LLC
Albena Kantardzhieva: Casebia Therapeutics LLC
Shailendra Sane: Casebia Therapeutics LLC
Karolina Kosakowska: Casebia Therapeutics LLC
Brian Cafferty: Casebia Therapeutics LLC
Jan Tebbe: Bayer AG
Sarah J. Spencer: CRISPR Therapeutics INC
Scott Munzer: Casebia Therapeutics LLC
Christopher J. Cheng: Casebia Therapeutics LLC
Abraham Scaria: Casebia Therapeutics LLC
Andrew M. Scharenberg: Casebia Therapeutics LLC
André Cohnen: Bayer AG
Wayne M. Coco: Bayer AG

Nature Communications, 2021, vol. 12, issue 1, 1-12

Abstract: Abstract Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a “GG” dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.

Date: 2021
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DOI: 10.1038/s41467-021-24454-5

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