Establishment of a highly efficient method for β-thalassemia CD17 (A &gt; T) point mutation in HEK293T cell line

doi:10.13418/j.issn.1001-165x.2022.5.14

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Chinese Journal of Clinical Anatomy ›› 2022, Vol. 40 ›› Issue (5) : 581-586. DOI: 10.13418/j.issn.1001-165x.2022.5.14

Establishment of a highly efficient method for β-thalassemia CD17 (A > T) point mutation in HEK293T cell line

Liu Yongxiang^1,2, Cai Bing², Xu Yan², Zeng Yanhong², Zhou Shaohu¹, Mai Qingyun²*

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Abstract

Objective To establish an efficient method for constructing HEK293T cell line of β-thalassemia CD17 (A>T) point mutation. Methods Using a modified CRISPR/Cas9 gene editing technology, termed ‘CORRECT’ (consecutive re-Guide or re-Cas steps to erase CRISPR/Cas-blocked targets) for scarless genome editing. Firstly, the cleavage of HBB gene in HEK293T cells was induced by electro-transfection of CRISPR/Cas9 plasmid. Then, single-stranded oligo DNA nucleotides (ssODNs) with CD17 (A>T) point mutation and synonymous mutation (G>T) were used as homologous templates for repair. The HEK293 T cell line with β-globin CD17 (A>T) point mutation was obtained by monoclonal screening and sequencing analysis. Results A HEK293T cell line with point mutation of β-thalassemia CD17 (A>T) genotype was successfully obtained by 'CORRECT' technique. The introduction of synonymous mutation might reduced re-editing of Cas9 protein to the target, which greatly improved the efficiency of single base mutation. Conclusions The point mutation HEK293T cell line can be efficiently obtained by 'CORRECT' technique, which is of great significance for the establishment of single base mutation cell lines and animal models.

Key words

β-thalassemia； / / CRISPR/Cas9； / / Synonymous mutation； / / Single base mutation

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Liu Yongxiang, Cai Bing, Xu Yan, Zeng Yanhong, Zhou Shaohu, Mai Qingyun. Establishment of a highly efficient method for β-thalassemia CD17 (A > T) point mutation in HEK293T cell line[J]. Chinese Journal of Clinical Anatomy. 2022, 40(5): 581-586 https://doi.org/10.13418/j.issn.1001-165x.2022.5.14

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