Precise gene targeting (GT) still remains a bottleneck in plant biotechnology and crop improvement, despite the enormous transformative impact of CRISPR-Cas nucleases.GT harness the endogenous double-strand repair pathways; however, these pathways and factors influencing them are poorly understood in plants. Here, a comprehensive study was carried out by combining physiological, molecular, and genome-editing approaches; the study provides novel insights into the complex role of DNA repair factors beyond maintaining genome integrity. Phenotypic analyses of multiple mutants showed that genes traditionally associated with DNA repair also influence plant growth, which was measured in parameters such as height, root length, and seed size. This is evidenced by significant growth defects in mre11 mutants and declined reproductive fitness in fanci mutants. Thus, these observations suggest that DNA repair genes function in connection with the intersection of genome maintenance, developmental regulation, and fertility. The transcriptomic profiling of these mutants further supported this conclusion by showing the altered expression of genes involved in growth, development, and stress response. This study has also characterised the role of several DNA repair genes, such as RAD5A, EXO1, FANCI, and BRCA1, to influence GT outcome to a variable extent. To define the role of these and other selected DNA repair genes, CRISPR-based mutational profiling was carried out, which revealed locus-specific effects on repair outcomes and choice of repair pathway, with proteins such as FANCD2, EXO1, RAD5A, WRN, FANCI, BRCA1 and XRCC2 displaying variable influences on single-strand annealing (SSA) and/or non-homologous end joining (NHEJ). Notably, the involvement of EXO1a/1b, XRCC2, BRCA1 and FANCI in NHEJ was unexpected, as these proteins are more commonly associated with homologous recombination (HR) or the Fanconi anaemia (FA) pathway. Among the mutants studied, Exo 1 seems to be involved in an SSA-like mechanism to repair induced DSB. Analysis of fanci mutant, a Fanconi anaemia pathway component, revealed its functional conservation and involvement in mitigating replication stress along with Mus81 in a non-redundant pathway. The above findings will not only advance our understanding of fundamental plant DNA repair mechanisms but also inform strategies to enhance gene targeting for crop improvement.
