The recent development of genome editing technologies using CRISPR/Cas9 has marked a new era in precise genome editing. This study reports on genome editing using seven single-guide RNAs (sgRNAs) to target different positions in the OsGTL1 promoter in rice (Oryza sativa L.). This research focused on identifying CRISPR-edited plants before evaluating the effects of OsGTL1 modification in rice progeny. We successfully developed gene-edited lines that have different types of mutations. Moreover, our study detected at least 35% of genetic chimerism in the first generations of the edited lines. This chimerism resulted in a mosaic of genotypes within an individual plant, suggesting multiple CRISPR-Cas9 events targeting the target region. We successfully identified transgene-free lines in later generations. Various modifications occurred in the targeted region, including large and small deletions, single-base insertions and deletions, and inversions. This study highlights the complex challenges and opportunities of applying CRISPR/Cas9 to improve rice traits, emphasizing the importance of detailed characterization of edited lines to fully understand a gene's function and its pleiotropic effects.
