Development And Evaluation Of Endosens Cell Model For Skin Sensitization Prediction Based On CRISPR/Cas9 Technology

Skin sensitization testing is an important part of safety assessment for cosmetic ingredients and product.With concerns of the animal welfare and 3R principles,especially as the animal testing has been baned for cosmetic products and their ingredients in the EU,the development of in vitro alternative methods for cosmetics safety assessment has become an international trend and research focus.The skin sensitization adverse outcome pathway is the key steps to develop in vitro alternative methods.Currently,the reporter cell models that mimic keratinocyte activation,KeratinoSens^TM and LuSens,are based on Keap1-Nrf2 mimic induction by conducting antioxidant response element-luciferase plasmids.These have been proved to be feasible test methods for skin sensitization in vitro.However,plasmid-based reporters may ignore comprehensive aspects of induction,containing uncertainty for the hazard identification.Because of the development of CRISPR/Cas9 in recent years,it appears that precise insertion of a reporter gene into the endogenous gene frame would allow synchronous expression between the endogenous gene and reporter gene,and would much closely mimic the state of cells.In this study,we developed a novel HaCaT-based reporter system,EndoSens,in which Luciferase gene was specifically inserted into the cassette of heme oxygenase decycling1?HMOX1?by CRISPR/Cas9.Thus,luciferase activity within the novel cell line actually reflected the expression of the endogenous HMOX1 gene.The EndoSens assay was evaluated with 35 substances,including 20 coded test substances.Testing data from 20 coded substances showed an accuracy of 90.0%,sensitivity of 91.7%,and specificity of 87.5%,which are over the OECD basic requirement.Among the 35 chemicals examined,the predictivity was better than that of the validated KeratinoSens^TM.Furthermore,The EndoSens also accurately predicted the skin sensitization of plant extract,such as cinnamon extracts.In conclusion,the EndoSens assay could be successfully applied for estimating the skin sensitization potential of chemicals and complex plant extract.The cell model EndoSens based on the CRISPR/Cas9 has been provided with independent intellectual property rights and would help the development of in vitro skin sensitization testing in our country as in the frond of cosmetics safety assessment.