A rapid CRISPR-Cas12a/T7EI integrated workflow for high-throughput screening of homozygous mutant cell lines

Abstract

Efficient screening for homozygous mutant cell lines, particularly those resulting from low-efficiency CRISPR-Cas9 editing, remains challenging. Here, we developed HomoSelect-CT, an integrated workflow combining CRISPR-Cas12a nucleic acid detection with T7 Endonuclease I (T7EI) genotyping, designed to streamline the screening process for homozygous mutant cell lines. This method requires no specialized instrumentation, enhancing accessibility and efficiency. We validated HomoSelect-CT by successfully identifying homozygous mutants in CRISPR-Cas9-edited THP-1 cells, which was confirmed by Sanger sequencing and Western blot (WB). These findings demonstrate that HomoSelect-CT is a robust and efficient alternative for the rapid isolation of genome-edited cell lines. The entire screening workflow, from monoclonal cultures to confirmed homozygous mutants, is completed in under 4 h, requiring only standard PCR equipment and routine reagents. Thus, HomoSelect-CT represents a significant advancement in CRISPR screening methodology, offering remarkable simplicity and enabling high-throughput screening that is particularly suitable for mutants arising from low-efficiency editing events.