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Abstract: Digestive enzymes of fish are critical to food digestion at the larval stage, but convincing evidence proving the function and necessity of the associated digestive enzymes remains lacking. In this study, we generated the trypsin gene (try) and amylase gene (amy) in the japanese medaka (Oryzias latipes) using CRISPR for the first time. try deletion significantly decreased the expression of try and digestive capacity in try^-/- medaka larvae; after 8 h digestion, incompletely digested brine shrimp was observed in the digestive tract at 4 and 15 days post-hatching (dph) of try^-/- medaka larvae. Furthermore, the height of intestinal villi and total body length decreased significantly within 15 dph try^-/- medaka larvae. However, amy deletion did not influence the digestion of medaka larvae at 4 dph. Only a small amount of incompletely digested brine shrimp was observed in 15-dph amy^-/- medaka larvae. Further analysis of the growth, nitrogen metabolism, and intestinal microbes of try^-/- adult medaka showed that the body length and weight of adult medaka decreased significantly, while the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the blood significantly increased. Pathological observation of liver and intestinal tissues showed that try knockout resulted in vacuolar degeneration of liver cells, thinning of the intestinal wall, sparse arrangement of villi, and lower villi height. High-throughput 16s rRNA sequencing revealed that try knockout reduced the diversity of intestinal microbes. These findings demonstrated that try was indispensable for medaka larvae because it continuously affects their growth, nitrogen metabolism, and intestinal development.