目的 利用优化的CRISPR/Cas9（Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR）技术，高效建立一种通过递送新型Cas9-gRNA核糖核蛋白复合物（RNP）制备人类重组激活基因1（RAG1）c.946T>G点突变的前B淋巴细胞系的方法，并对突变体进行功能分析。 方法 实验以人前B淋巴细胞系（Nalm6细胞）为研究对象，在体外将化学修饰的gRNA（guide RNA）和高保真HIFI Cas9蛋白孵育形成RNP复合物，通过细胞核转染技术将RNP和含RAG1（T>G）目标突变与同义突变（C>A）的超分子单链寡核苷酸（Ultramer-ssODNs）同源模板带入细胞内，诱导RAG1基因发生切割与重组，T7E Ⅰ 酶切检测基因编辑效率，并经计数稀释法进行单克隆筛选，Sanger测序鉴定基因型。 结果 成功构建RAG1（c.946T>G）纯合点突变的细胞系，切割效率高达78.21%，单克隆筛选获得较高比例33.33%（4/12）的目标单克隆。通过功能实验发现c.946T>G点突变导致前B淋巴细胞RAG1、RAG2蛋白表达下调，细胞凋亡比例增多，可能是导致最终成熟B淋巴细胞的生成减少的原因。 结论 该研究通过优化的Cas9 RNP技术高效获得RAG1基因c.946T>G纯合点突变的前B淋巴细胞系，为单碱基突变疾病模型的建立提供实验依据，更为提升剪切精确性、提高转染效率提供了新的方法。
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.
关键词
原发性免疫缺陷； /
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前B淋巴细胞； /
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核糖核蛋白复合物； /
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RAG1
Key words
Primary immunodeficiency； /
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Pre-B lymphocyte； /
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Ribonucleoprotein complex； /
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RAG1
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基金
广东省医学科研基金项目(A2023401)；国家中医药传承创新中心科研专项青年项目(2023QN02)；广东省中医药局科研项目(20241103)
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