The fatty acids content of castor (Ricinus communis L.) seed oil is 80-90% ricinoleic acid, which is a hydroxy fatty acid (HFA). The structures and functional groups of HFAs are different from those of common fatty acids and are useful in various industrial applications. However, castor seeds contain the toxin ricin and an allergenic protein, which limit their cultivation. Accordingly, many researchers are conducting studies to enhance the production of HFAs in Arabidopsis thaliana, a model plant for oil crops. Oleate 12-hydroxylase (FAH12) from castor (RcFAH12), which synthesizes HFA (18:1-OH), was transformed into an Arabidopsis fae1 mutant, resulting in the CL37 line producing 17% maximum HFA. In addition, phospholipid diacylglycerol acyltransferase 1-2 (RcPDAT1-2), which catalyzes the production of triacylglycerol by transferring HFA from phosphatidylcholine to diacylglycerol, was transformed into the CL37 line to develop a P327 line that produces 25% HFA. In this study, we investigated changes in HFA content when endogenous Arabidopsis PDAT1 (AtPDAT1) of the P327 line was edited using the CRISPR/Cas9 technique. Successful mutation resulted in three independent lines with different mutation patterns, which were transmitted until the T4 generation. Fatty acid analysis of the seeds showed that HFA content had decreased in all three mutant lines. This finding indicates that AtPDAT1 and RcPDAT1-2 in P327 line are involved in the transfer of HFAs to triacylglycerol.