A start codon-targeted genome editing strategy for generating hypomorphic mutants of lethal plant genes

In plants, the functional characterization of essential genes is often hindered by the lethality associated with complete loss-of-function alleles. Here, we present a genome editing-based strategy to generate viable hypomorphic alleles through selective removal of the translation start codon. Using Arabidopsis thaliana NON-SMC ELEMENT 1, which encodes a conserved component of the Structural Maintenance of Chromosomes (SMC) 5/6 complex involved in DNA repair and genome stability, as a model, we generated CRISPR-Cas9-edited alleles lacking the start codon. These homozygous mutants exhibited severe developmental defects, including stunted growth and failure to form true leaves. Moreover, they displayed molecular hallmarks of genome instability, such as increased DNA fragmentation, upregulation of DNA repair and cell cycle checkpoint genes, and root meristem cell death. Complementation assays using wild-type and mutated NSE1 genomic constructs confirmed that these alleles retained partial gene function. Overall, the use of start codon removal as an editing strategy is a robust and broadly applicable approach for generating hypomorphic alleles without relying on transcript-level manipulations, such as RNAi. This work therefore provides a practical demonstration of a novel editing strategy for dissecting the functions of essential genes that are otherwise genetically intractable. Consequently, this approach expands the functional genomics toolkit and opens new avenues for basic plant biology and advanced biotechnological applications.