KOnezumi-AID: A Software to Automate the Design of Multiplex Knockouts Using Target-AID

With the groundbreaking advancements in genome editing technologies, particularly CRISPR-Cas9, creating knockout mutants has become highly efficient. However, the CRISPR-Cas9 system introduces DNA double-strand breaks, which can increase the risk of chromosomal rear-rangements, presenting a major obstacle when attempting to knockout multiple genes simultaneously. Base editing systems like Target-AID provide a safer alternative by enabling precise base modifications without requiring DNA double-strand breaks, making them a promising solution for addressing this challenge. Nevertheless, the absence of adequate tools to support Target-AID-based gene knockouts highlights the need for a comprehensive system to design guide RNA (gRNA) for the simultaneous knockout of multiple genes. Here, we present KOnezumi-AID, a command-line tool designed to facilitate gRNA design for Target-AID-mediated genome editing. KOnezumi-AID supports gene knockout strategies by inducing premature termination codons or promoting exon skipping. It generates experiment-ready gRNA designs for both mouse and human genomes. Additionally, KOnezumi-AID offers batch processing capabilities, enabling rapid and precise gRNA design for large-scale genome editing projects such as CRISPR screening. In summary, KOnezumi-AID provides an efficient and user-friendly tool for gRNA design, streamlining genome editing workflows and advancing gene knockout research.