| Alpha-thalassemia is an autosomal single-gene inherited disease.It is a chronic hemolytic anemia caused by genetic mutations that cause abnormal globin structure and abnormal hemoglobin(Hb)composition.SEA-type ?-thalassemia is the most prevalent type of ?-thalassemia.Its genetic feature is that both copies of the ?-globin gene on one chromosome are missing.Hb Bart's hydrops fetalis is a serious lethal disease in ?-thalassemia.When both spouses are SEA-type ?-thalassemia patients,there is a 25%probability that the fetus is Hb Bart's hydrops fetus,which not only causes the fetus to fail to survive,but also has a greater threat to the life of the pregnant woman.Therefore,the detection of SEA-type ?-thalassemia and the prenatal detection of their fetuses should be strengthened.Early detection methods for SEA-type thalassemia include complete blood count,high performance liquid chromatography and hemoglobin electrophoresis.In the past 30 years,a variety of molecular genetic testing methods,such as southern blot and DNA sequencing,can accurately detect the variation of most ?-globin genes,thereby diagnosing ?-thalassemia.However,these methods are not suitable for the diagnosis of all cases of ?-thalassemia due to their cumbersome operation and high cost.Rapid screening methods such as gap-PCR have been widely used in the detection of seven common deletion type?-thalassemia.In order to ensure the accuracy of the test results,the laboratory needs to verify the performance of commercial reagents before routine testing,and the manufacturer needs to use positive samples for validation when developing methods.In daily testing,laboratories should carry out internal quality control,participate in external quality assessment/proficiency testing,and these processes are inseparable from quality control materials.Common genetic disease detection quality control materials can be divided into the following three categories:firstly,non-cellular quality control materials,such as synthetic plasmids;secondly,immortalized cell lines constructed using patient cells;thirdly,cell lines obtained by introducing the target mutation on the basis of normal cell lines through gene editing technology.However,the first two types of quality control materials have certain limitations,such as the lack of extraction process of genomic DNA in non-cellular quality control materials,and immortalized patient cell quality control materials contain fewer mutation types.While the quality control materials prepared by gene editing technology can not only contain more mutation types,but also simulate real samples.At present,CRISPR/Cas9 gene editing technology has been applied to the preparation of quality control materials,but the cell lines used in these studies are adherent cell lines,and there is no study on the preparation of quality control materials using lymphoblastoid cell lines,while the parent-child cell lines in internationally recognized mother/father/son(GM 12878/GM12877/GM12884)normal family(CEPH/UTAH PEDIGREE 1463)are all lymphoblastoid cell lines.Therefore,the construction of mutant cell lines with CRISPR/Cas9 gene editing technology in lymphoblastoid cell line is conducive to the construction of the same type of mutation in internationally recognized parent-child cell lines,so that not only quality control materials for detection of SEA-type?-thalassemia can be prepared,but also matched maternal and fetal cell free DNA can be produced by pairing of parent-child cell lines and enzymatic digestion.Cell free DNA can lay a foundation for the preparation of quality control materials for non-invasive prenatal testing of ?-thalassemia in the future.In this study,we used GM12878 cell line stably expressing Cas9 as the basic cell line.According to the characteristics of 19kb DNA fragment deletion on chromosome 16 in SEA-type ?-thalassemia,two different sgRNAs were designed for the two break sites of the deleted fragment on chromosome 16,and the sgRNAs were in vitro transcribed and purified on the base of recombinant pX458 plasmid.Purified sgRNA was introduced into GM 12878 cell line stably expressing Cas9 protein by electrotransfection to prepare cell line with SEA-type ?-thalassemia gene mutation.We also conducted a study of direct transfection of recombinant pX458 plasmids(expressing Cas protein and sgRNA)into a common GM 12878 cell line,and compared the editing efficiency between them.The final edited cell lines were verified by gap-PCR,Sanger sequencing and whole genome sequencing.The results showed that electrotransfection of purified in vitro transcribed sgRNA into GM 12878 cell line stably expressing Cas9 protein was a more effective gene editing method than direct electrotransfection of recombinant pX458 plasmid to common GM12878 cell line.The electrophoretic results of the edited monoclonal cells verified by gap-PCR showed that the electrophoretic band appeared at the expected position,which was the same position as the electrophoretic band of SEA-type ?-thalassemia.The PCR amplification products of monoclonal cells with positive gap-PCR validation were verified by Sanger sequencing,and the results showed that a 19kb deletion mutation was formed on chromosome 16,which was the same as the deletion position of the SEA-type ?-thalassemia gene mutation.However,there are short insertion and deletion mutations of about 10bp at the break site,which is negligible compared with the nearly 19kb deletion of DNA fragments in the SEA-type ?-thalassemia gene mutation.The results of whole genome sequencing analysis also showed a concordance with the SEA-type ?-thalassemia gene mutation.In summary,we prepared cell lines containing SEA-type ?-thalassemia mutation by electrotransfecting transcribed in vitro sgRNA into GM12878 cell line stably expressing Cas9 protein,and validated them by different methods.The results show that this method is a more effective way of gene editing.At the same time,the validation results of different methods also showed that GM 12878 cell line containing SEA-type ?-thalassemia mutation after gene editing could be used as a quality control product for the detection of SEA-type?-thalassemia.The innovations of this study are as follows:(1)CRISPR/Cas9 gene editing technology is applied to the preparation of quality control products for genetic disease detection by electrotransfection-based method for the first time.(2)The cell lines used in this study are maternal cell lines derived from internationally recognized normal families,and the same type of mutations can be constructed in fetal cell lines derived from normal families by the same method,thus providing a material basis for the preparation of quality control products for noninvasive prenatal testing of ?-thalassemia,which is also the research content considered in the next stage of this study. |
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