Integrative non-viral vectors such as transposons engineered to mediate targeted gene transfer into safe harbor sites in the genome may be a promising approach for the production of therapeutic proteins or for gene therapy in an efficient and secure way. In this context, we designed and evaluated two strategies for targeting the nuclear ribosomal DNA (rDNA) loci. One approach relied on the co-location of the transposase and transposon near transcriptionally active rDNA copies using a nucleolar localization signal (NoLS). Another one consisted of targeting the 18S-coding region in the rDNA loci using a NoLS-FokI-dCas9 endonuclease to perform targeted transgene knock-in. We show that integration into the rDNA of Chinese hamster ovary (CHO) cells can be achieved at a high frequency using the piggyBac transposon system, indicating that the rDNA is highly accessible for transposition. Consistently, rDNA-targeted transposition events were most frequently obtained when both the piggyBac transposon DNA and the transposase were nucleoli-targeted, yielding cells displaying stable and homogeneous expression of the transgene. This approach thus provides an alternative strategy to improve targeted transgene delivery and protein expression using CHO cells.