Efficient establishment of human pre-B lymphocyte RAG1 c.946T&gt;G point mutation cell line using optimized Cas9 RNP technology and its function analysis

doi:10.13418/j.issn.1001-165x.2025.2.11

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Chinese Journal of Clinical Anatomy ›› 2025, Vol. 43 ›› Issue (2) : 175-182. DOI: 10.13418/j.issn.1001-165x.2025.2.11

Efficient establishment of human pre-B lymphocyte RAG1 c.946T>G point mutation cell line using optimized Cas9 RNP technology and its function analysis

Liu Yongxiang¹, Liu Caifeng², Li Zishuo¹, Li Yunshi¹, Chen Xingmei¹, Zhou Na¹, Zhou Shaohu¹*, Huang Xuekun¹*

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Abstract

Objective Using enhanced CRISPR/Cas9 technology (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR), to construct an efficient method for producing a human pre-B lymphocyte line with the recombination activating gene 1 (RAG1) c.946 T>G point mutation by delivering a novel Cas9-gRNA ribonucleoprotein (RNP) complex and investigate the mutant's function. Methods Human pre-B lymphocyte cell line (Nalm6 cell) was employed as the research subject in this study. RNP complexes were generated in vitro by combining high-fidelity HIFI Cas9 protein with chemically modified gRNA (gRNA). Using nuclear transfection technique, RNP and ultramer single-stranded oligodeoxynucleotides (Ultramer-ssODNs) homologous templates containing RAG1 c.946T>G target mutation and synonymous mutation (C>A) were introduced into cells to induce RAG1 gene cleavage and recombination. The gene editing efficiency was detected by T7E I digestion assay, and monoclonal screening was performed by counting dilution method. Genotypes were identified by Sanger sequencing. Results Ultimately, the RAG1 (c.946T>G) homozygous point mutation cell line was successfully constructed, and the cleavage efficiency reached 78.21%. Additionally, a high percentage of 33.33% (4/12) of the desired monoclonal was acquired using monoclonal selection. Preliminary functional investigations revealed that the c.946T>G point mutation decreased expression of RAG1 and RAG2 proteins in pre-B cells and increased apoptosis rate, which could explain the decrease in the generation of mature B lymphocytes. Conclusions This study's optimized Cas9 RNP technology effectively produce the RAG1 c.946T>G homozygous point mutation in the pre-B lymphocyte line, providing experimental basis for the creation of a single base mutation disease model as well as a novel technique for enhancing edit accuracy and improving transfection efficiency.

Key words

Primary immunodeficiency； / / Pre-B lymphocyte； / / Ribonucleoprotein complex； / / RAG1

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Liu Yongxiang, Liu Caifeng, Li Zishuo, Li Yunshi, Chen Xingmei, Zhou Na, Zhou Shaohu, Huang Xuekun. Efficient establishment of human pre-B lymphocyte RAG1 c.946T>G point mutation cell line using optimized Cas9 RNP technology and its function analysis[J]. Chinese Journal of Clinical Anatomy. 2025, 43(2): 175-182 https://doi.org/10.13418/j.issn.1001-165x.2025.2.11

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