In vivo evaluation of guide-free Cas9-induced safety risks in a pig model

The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy.However,there are concerns about the safety of this system,specifically related to the use of guide-free Cas9.Previous studies have shown that guidefree Cas9 can induce genomic instability in vitro.However,the in vivo safety risks associated with guide-free Cas9 have not been evaluated,which is necessary for the development of gene therapy in clinical settings.In this study,we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo.Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo.The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein.Moreover,prolonged expression of Cas9 in pigs led to abnormal phenotypes,including a significant decrease in body weight,which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction.Furthermore,we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression,raising the risk of tumor occurrence.Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy.This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRiSPR/Cas9 system in clinical practice.