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Mucopolysaccharidosis type I (MPS I) is caused by alpha-L-iduronidase enzyme (IDUA) deficiency and lysosomal accumulation of glycosaminoglycans (GAGs). The accumulation of GAGs affects important cellular processes and triggers a cascade of responses altering cellular homeostasis and leading to multisystemic dysfunctions. In this disease, the bone/joint complications reduce mobility. Current treatments do not correct these complications. Therefore, it is necessary to look for new therapies. An alternative is gene editing using the CRISPRCas9 system. This work aims to evaluate the efficiency of cationic liposomes carrying plasmids with the CRISPRCas9 system plus a donor vector to replace IDUA in fibroblast-like synoviocytes. MPS I mouse fibroblast-like synoviocytes (FLS) were transfected with 1) Liposome/CRISPR+Donor IDUA or 2)Liposome/Donor IDUA or 3)Lipofectamine 3000® Lipofectamine/Donor IDUA or 4)Lipofectamine/CRISPR+Donor IDUA. The cells were maintained in culture and collected at 4 times (3, 7, 15, and 30 days) after transfection. IDUA activity and total proteins were measured at each of these times. The FLS from untreated MPS I and from normal mice were cultured in the same conditions and used as controls. The incubation of the complexes with FLS demonstrated high cell viability (over 70%) and no statistical difference (p >0.05) between treatments, and these results showed that the Liposome presented low cytotoxicity. At all times, the untreated FLS MPS I showed very low levels of IDUA. The treatment with Lipofectamine/CRISPR+Donor IDUA and Liposome/CRISPR+Donor IDUA always showed a significant increase in IDUA production (p < 0.0001 and p <0.001). This increase was sustained for up to 30 days. The treatment with Lipofectamine/CRISPR+Donor IDUA reached an average of 4.0% (±0.26), of IDUA activity found in normal FLS, while the treatment with Liposome/CRISPR+Donor IDUA reached an average of 2.8% (±0.28). Both treatments were not significantly different from each other (p >0.05). Despite the enzyme levels not being normalized, due to cross-correction (enzyme exchange) between neighboring cells, an increase of 1% in the production of the enzyme could be enough to reduce the accumulation of glycosaminoglycans in lysosomes. This study demonstrates that the Liposome is appropriate for gene editing studies. New studies are being conducted in MPS I mice in vivo. [ABSTRACT FROM AUTHOR]