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Utilizing CRISPR/Cas9 technology to prepare lymphoblastoid cell lines harboring genetic mutations for generating quality control materials in genetic testing

BACKGROUND: To meet the requirements of the rapidly progressing genetic testing technologies in clinical laboratories, assuring the quality of genetic tests by utilizing appropriate quality control materials is of paramount importance. The CRISPR/Cas9 technology was used to prepare quality control m...

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Detalles Bibliográficos
Autores principales: Zhou, Li, Li, Rui, Zhang, Runling, Peng, Rongxue, Chen, Kun, Gao, Peng, Zhang, Jiawei, Zhang, Rui, Li, Jinming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370731/
https://www.ncbi.nlm.nih.gov/pubmed/32118319
http://dx.doi.org/10.1002/jcla.23256
Descripción
Sumario:BACKGROUND: To meet the requirements of the rapidly progressing genetic testing technologies in clinical laboratories, assuring the quality of genetic tests by utilizing appropriate quality control materials is of paramount importance. The CRISPR/Cas9 technology was used to prepare quality control materials because genome‐edited human cell lines are one of the major resources for quality control materials. METHODS: In this study, in vitro transcribed sgRNA were transfected into a Cas9‐expressing lymphoblastoid cell line (LCL)—by electroporation—to simulate the SEA‐type deletion observed in α‐thalassemia. The edited positive cell line was screened and identified by polymerase chain reaction (PCR) followed by Sanger sequencing. The whole‐genome sequencing was also performed to show evidence of predicted mutation. RESULTS: The results showed that electroporation of the in vitro transcribed gRNAs into stable Cas9‐expressing LCL was a more efficient gene‐editing technique as compared to plasmid‐mediated transfection, and that the positive rates could reach up to 35.9%. The predominance of indel sizes relative to the predicted deletion length was clustered between 10 and 0 bp. The results of whole‐genome sequencing also demonstrated the existence of SEA‐type deletion of α‐thalassemia. CONCLUSIONS: Gene‐editing based on Cas9‐expressing LCL by electroporation of sgRNA was a more efficient approach to introduce mutations for generating quality control materials for genetic testing. The edited lymphoblastoid cell lines were feasible to serve as quality control materials in genetic testing.