A Novel DNMT3A R882H Cell Line Generated Using CRISPR/Cas9 Technology as a Universal Quality Control Material for Clinical Testing in Acute Myeloid Leukemia
10.13865/j.cnki.cjbmb.2021.07.1228
- Author:
Ning WANG
1
;
Ya-Li WEI
1
;
Rui ZHOU
1
;
Xu AN
2
;
Qin-Mei CAO
3
Author Information
1. Department of Clinical Laboratory, Beijing Chaoyang Hospital, The Third Clinical Medical College of Capital Medical University
2. Department of Clinical Laboratory, Tongzhou Maternal and Child Health Hospital of Beijing
3. Department of Clinical Laboratory, China-Japan Friendship Hospital, Clinical Medical College of Capital Medical University
- Publication Type:Journal Article
- Keywords:
acute myeloid leukemia (AML);
CRISPR/Cas9;
next-generation sequencing;
quality control materials;
sanger sequencing
- From:
Chinese Journal of Biochemistry and Molecular Biology
2021;37(8):1118-1123
- CountryChina
- Language:Chinese
-
Abstract:
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.
