Gene knock-in (KI) technology holds great promise in cell therapy and industrial biomanufacturing. However, conventional knock-in strategies often suffer from low efficiency, limiting their broader application in these fields. Current knock-in methods largely depend on homology-directed repair (HDR), but cells naturally favor non-homologous end joining (NHEJ) to repair double-strand breaks (DSBs), which leads to low integration efficiency. Among the existing platforms, CRISPR/HDR combined with AAV (adeno-associated virus) donor systems is frequently used and relatively efficient. However, AAV donors are prone to tandem insertions into the genome and come with limitations such as high cost, complex production, and limited cargo capacity. These challenges further restrict the widespread application of gene knock-in technologies.

HES-KI is a novel knock-in platform that targets the C-terminal exon region of essential genes for CRISPR editing. A knock-in template is designed such that successful integration restores the function of the essential gene and inserts the desired transgene. In contrast, cells with failed knock-ins—such as those repaired via NHEJ resulting in indels—lose the function of the essential gene and cannot survive. This creates a natural selection pressure that enriches for successfully edited cells.