对于不同基因位点的突变,急性髓系白血病的发病机制和治疗策略各不相同,因此精准的分子检测在急性髓系白血病的诊断中发挥至关重要的作用.但是目前临床实验室进行分子检测时,缺乏高度模拟病人样本的室内质控品,同时也无完善的外部质量评估系统来评估临床实验室检测结果的正确性.为了提高临床实验室分子检测的正确性和可信度,我们使用C RISP R/Cas9技术构建DNMT3A(R882H,2645G＞A)HEK293T细胞系,以用于急性髓系白血病分子检测的质量控制.同时将单链供体模板中cas9蛋白识别位点AGG更换为AGA,以阻止同源重组的细胞系再次被cas9蛋白切割,从而提高同源重组细胞系制作的成功几率.经验证构建成功的DNMT3A(R882H,2645G＞A)细胞系能够稳定遗传,同时使用该细胞系DNA提取产物制作的室间质评样本中目的基因的突变频率,在2种Sanger测序仪器和3种高通量测序仪器上得到较好的验证.以上结果表明,制备的DNMT3A4(R882H,2645G＞A)HEK293T细胞系的DNA提取产物不仅可用作室内质量控制,同时也能作为监控不同厂家、多种平台的室间质评样本,从而提高临床实验室分子检测的准确性和可信度.“,”The pathogenesis and treatment strategies of acute myeloid leukemia(AML)are different for disparate gene mutations.Therefore,precise molecular testing plays a vital role in its diagnosis.However,when clinical laboratories perform molecular testing,internal quality control materials similar to clinical samples for molecular testing are lacking.At the same time,there is no related external quality assessment system to evaluate clinical laboratory test results.In order to improve the accuracy and credibility of molecular testing in clinical laboratories,we used the CRISPR/Cas9 technology to construct the DNMT3A(R882H,2645G＞A)HEK293T cell line for the quality control of AML molecular testing.We replaced the cas9 protein recognition site AGG in the ssODN with AGA to prevent the homologous recombination cell line from being cleaved by the cas9 protein again,thereby increasing the success rate of homologous recombination cell line production.It has been verified that the DNMT3A(R882H,2645G＞A)cell line can be inherited stably.The mutation frequency of the external quality assessment sample made by the DNA extraction of DNMT3A(R882H,2645G＞A)HEK293T cell line was very stable,tested by two Sanger sequencing instruments and three NGS instruments.The results above showed that the DNA extraction of DNMT3A(R882H,2645G＞A)HEK293T cell line can not only be used as internal quality control,but also be used as external quality assurance samples for monitoring different manufacturers and platforms,thereby improving the accuracy and credibility of molecular testing in clinical laboratories.