Systematic Tiling Approach for Engineering Smaller Genome Editing Tools

Although current versions of various proteins that exist in nature may be sufficient for organismal survival, all may not be readily transferable for therapeutic purposes without further engineering or tweaking of native sequences. The most recent and popular example within the genetic engineering field is the CRISPR/Cas9 system. Following its widespread adoption as a programmable tool for genome editing, the nuclease-null ‘dead’ Cas9 variant has been repurposed by fusing it to effector domains (1-3). Such effectors have enabled unprecedented ease of eukaryotic transcriptome and epigenome manipulation (4-6). As a result, these tools are now highly valued due to their easily usable nature and promising potential for functional and therapeutic studies (7). However, clinical application and delivery has been hindered due to the large protein sizes of these tools. Without the ability to effectively and safely deliver such vectors to patients, these tools will never reach their full therapeutic potential. To this end, we perform a systematic tiling approach using a library-scale Cas9-based method to identify dispensable amino acid stretches for efficient Cas9 function with the goal of identifying a functional “miniCas9” version. This approach allows one to address a central question of the limit to which one can tweak nature’s existing forms of proteins to fit them, via size, to standard delivery vectors for therapeutic purposes.

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