Enhancing Editing Efficiency, Versatility and Guide Design of CHyMErA Screening Platform

Division Of Basic Sciences - Nci

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Abstract

We have successfully developed a suite of cell lines expressing diverse Cas12a variants and conducted comprehensive functional screening using our Cas9-Cas12a CHyMErA platform, in conjunction with an optimized hgRNA library. These screens identified Cas12a variants exhibiting enhanced precision and editing efficiency compared to wild-type nucleases. Targeted validation experiments confirmed these improvements, and we have since deployed the superior variants in high-throughput screens aimed at identifying alternative exons that influence cellular fitness, as well as uncovering essential genes and pathways driving cancer cell proliferation. Beyond nuclease optimization, we conducted systematic screens to enhance and diversify the hgRNA scaffold, a critical component for enabling guide multiplexing and facilitating higher-order genetic interaction mapping. These efforts led to the identification of multiple scaffold variants that support efficient genome editing and are compatible with a range of phenotypic assays. In parallel, we are refining our guide design algorithms, with a particular focus on improving on-target scoring accuracy tailored to our enhanced Cas12a nucleases. This work aims to further increase the precision and effectiveness of genome editing. As part of this initiative, we compiled a comprehensive catalog of potential Cas9 and Cas12a target sites across the human and mouse genomes and systematically scored these sequences based on both on- and off-target characteristics. All associated data, including guide scores and genomic context, are being curated in an online database. We are actively developing a user-friendly, efficient search interface to support rapid design of individual or large-scale guide RNAs for both Cas9 and Cas12a platforms, thereby advancing a broad range of genome editing applications. Finally, we have established multiple CRISPRi and CRISPRa systems in human cell lines using either direct fusion or recruitment-based transcriptional modulator domains. The most promising platform were systematically assessed using high-throughput screening. Cells were transduced with an optimization library, and editing performance was assessed across various nuclease variants and modes of transcriptional repression. Our screens identified a preferred CHyMErA-CRISPRi system and highlighted the value of dual-targeting strategies in achieving stronger and more robust perturbation phenotypes. Additionally, we confirmed the suitability of different Cas12a guide scaffold variants for multiplexed CRISPRi applications.