基于CRISPR/Cas9技术的肿瘤突变负荷检测参考物质研究

肿瘤突变负荷（Tumor mutation burden, TMB）检测现已成为指导肿瘤免疫治疗的重要生物学标志物。目前临床上逐渐有越来越多的TMB检测方法被研发和使用，然而这些不同的检测、分析和计算方法常导致肿瘤患者TMB结果间存在差异，进而影响患者的下一步治疗方案。因此，为了保证TMB的临床检测有效性，研制可靠的且能模拟真实临床样本的TMB参考物质以确保其检测结果的准确性和一致性显得十分关键。我们前期的研究发现，特异性较差的CRISPR/Cas9系统可造成类似TMB的基因组多位点脱靶突变产生。故本研究拟采用CRISPR/Cas9基因编辑、异种移植和体外酶消化模拟细胞凋亡等技术，构建多种具有相同基因背景但含有不同TMB水平的肿瘤组织FFPE样本和血浆游离DNA样本参考物质，建立一种可长期持续生产、模拟真实肿瘤组织或血浆游离DNA特征的新型TMB检测参考物质构建平台。

The tumor mutation burden (TMB) is an important biomarker that plays a pivotal role in immunotherapeutic decision making for various kinds of cancer patients. Ensuring accuracy and reproducibility of TMB testing by different targeted next-generation sequencing panel requires reliable reference materials for monitoring assay sensitivity and specificity. Since our previous researches demonstrated that non-specific sgRNAs can lead to many unexpected mutations at any genomic loci after clustered regularly interspaced short palindromic repeat and the CRISPR-associated protein 9 (CRISPR/Cas9) editing in vitro. Hence, we are planning to develop a kind novel reference material by CRISPR/Cas9 technology for various kinds of TMB testing. In our project, CRISPR/Cas9 technology is going to be used to edit several novel cell lines which containing different mutations (high TMB level, medium TMB level, and low TMB level). After subcutaneous inoculation, the formalin-fixed, paraffin-embedded (FFPE) samples from xenografts can be prepared and tested for suitability as candidate reference materials for tumor tissue TMB testing. Besides, these constructed novel cell lines can also be digested by micrococcal nuclease (MNase) for the preparation of synthetic reference materials for cell-free DNA TMB testing. Without limitations on TMB level and production, our novel synthetic FFPE samples and cfDNA samples based on CRISPR/Cas9 editing, xenografts, and MNase digestion are suitable as candidate reference materials for the validation, verification, internal quality control, and proficiency testing of TMB detection. The successful development of these reference materials can provide substantial foundation for improving the quality of TMB research and ensuring the effective clinical application of TMB detection.