Making small genome editors more powerful– new paper published in Genome Biology
Genome editing therapies often rely on viral delivery systems such as adeno-associated viruses (AAVs), which have limited cargo capacity. Although RNA-guided nucleases from the Cas12f and TnpB families are attractive alternatives to larger enzymes such as spCas9, their relatively low editing activity in mammalian cells has restricted their broader application till now.
Do you need a super-active CRISPR nuclease for in vivo genome editing? Meet our two new compact editors with SpyCas9-like activity that are small enough for delivery in AAVs.
This exciting work was co-led by Fedor Gorbenko during his PhD and research assistant Iréne Sala, in collaboration with the Schwank lab (UZH) and the Siksnys lab (Vilnius University).
In this study, we developed a directed evolution platform using in a fluorescent reporter system in human cells to engineer compact genome editors with increased HDR efficiency. Through iterative rounds of mutagenesis and screening, we generated Cas12f1Super and TnpBSuper, two enhanced nucleases with up to an 11-fold increase in editing efficiency while maintaining high specificity. Moreover, the resulting variants also exhibit increased NHEJ activity. We further demonstrated efficient in vivo editing of the clinically relevant PCSK9 gene in mice following AAV delivery, with editing efficiencies of up to 61%. These results underscore the potential of Cas12f1Super and TnpBSuper for future therapeutic genome editing applications. When tested as a base-editor, the improved Cas12f1Super variant also substantially boosted the performance, achieving up to a tenfold increase in editing activity compared with the previously developed CasMINI.
By combining compact size with high activity, our Super genome editors broaden the genome engineering toolbox and open new opportunities for both basic research and therapeutic applications.
Congratulations to Fedor, Irene and all collaborators on this exciting achievement!
For more info, check out our new paper in Genome Biology!
