Improving editing efficiency and guide design of CHyMErA screening platform

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

We have cloned and established multiple cell lines for various Cas12a variants in the context of our CHyMErA screening Cas9/Cas12a screening platform. We have screened the different CHyMErA variants in multiple cell lines with a hgRNA library allowing us to compare editing efficiency as well as precision of the Cas12a variants in the context of our screening system. Our results revealed editing improvements with new Cas12a variants, which were confirmed with focused experiments. Genome-scale screens have been performed with the enhanced screening platform to map fitness exons and genes across various cancer cell lines. In addition to optimizing Cas nucleases we have also performed guide optimization screens in order to improve both the Cas12a direct repeat scaffold as well as to improve our guide design algorithms allowing us to achieve higher genome editing efficiency and precision. This screening data is currently being analyzed. In parallel we have been collecting all possible Cas9 and Cas12a target sites in the human and mouse genome, have scored guide sequences for on- and off-target properties and have uploaded guide sequences, genomic features and scores to an online data base. Current work focuses on making those data bases searchable in a time-effective fashion. The online tool will eventually allow for facile and efficient design of individual or large-scale guide design for Cas9 and Cas12a nuclease for numerous genome editing applications.