Construction Of Esophageal Squamous Cell Carcinoma Cell Models For Drug Resistance Using CRISPR/Cas SAM Library

According to estimates from the World Health Organization,cancer is the first or second leading cause of death before age 70 years in the most countries.Esophageal cancer ranks seventh in terms of incidence and sixth in mortality overall.Esophageal squamous cell carcinoma(ESCC)represents more than 90%of esophageal cancer cases in China.At present,the standard therapy for ESCC includes surgery,radiotherapy,and chemotherapy.Despite the use of multidisciplinary therapies,the prognosis of patients with ESCC remains poor.The overall survival rate at 5 years is only 20%.Resistance towards chemotherapy is a major obstacle in the treatment of ESCC.CRISPR/Cas system is a new gene editing technology in recent years.Benefiting from the simplicity and adaptability of CRISPR/Cas9,it opens the door for revealing gene function in biology and correcting gene defects in diseases.In 2015,Zhang synthesizes a library consisting of 70,290 guides targeting all human Ref Seq coding isoforms to screen for genes which,upon activation,confer resistance to a BRAF inhibitor.These results collectively demonstrate the potential of Cas9based activators as a powerful genetic perturbation technology,and designated it synergistic activation mediator(SAM).The project was intended to construct esophageal squamous cell carcinoma cell models for drug resistance using CRISPR/Cas SAM library.To develop a genome-scale transcription activation screening,KYSE30 cells and KYSE150 cells were transduced by the transcription activation system comprised by dCas-VP64、MS2-P65-HSF1、sgRNA(MS2),respectively.Finally,PCR analysis indicated that KYSE30 cells and KYSE150 cells stably integrated with SAM Cas9、effector components and SAM sgRNA libraries.Our work provided a bridge between foundational research and clinical treatment.