Written by丨XueyueCRISPR screens are powerful tools for discovering gene function. The study of different alleles of the same gene can provide unique insights into protein structure and functional mechanisms. Base editing technology is a CRISPR-based technology that introduces specific mutations in the guide sgRNA without double-strand breaks or homologous repair, is suitable for large-scale screening of allele functions, and can achieve single nucleotide resolution. T cells are an important target of immunity, but immunity has great limitations, and many patients are not reactive to it, so new methods are urgently needed to analyze specific nucleotide sequences in key genes and discover new functional genes. Base editing has been used for multiple knockouts of T cells, reducing the risk of chromosomal translocation, and has been applied in CAR T cell products. Using a high-throughput mutation screening platform can reveal a T cell allele profile that can be used to discover more target genes to improve immunity**. Recently, from the University of California, San Franciscoalexander marsonThe team is innaturePublished on the article entitledbase-editing mutagenesis maps alleles to tune human t cell functionsarticle. The studyThe newly developed base editing system is used to screen the function of alleles in primary T cells, providing a new tool for immunity**. 
To enable high-throughput use of base editors in human primary T cells, the authors optimized lentiviral delivery systems for the adenine base editor ABE8E (V106W) (abbreviated ABE) and the cytosine base editor EVOCDA1-BE4MAX (abbreviated CBE). In total, the authors designed a library containing 117,000 sgRNAs distributed in the coding regions of 385 genes associated with cell viability to identify mutations associated with cell activation. The authors assessed the editing efficiency of sgRNAs and defined sgRNAs that enhanced cell activation as positive and sgRNAs that inhibited cell activation responses as negative. The authors selected a subset of functional sgRNA corresponding targets from the screen. Analysis found that the system was able to identify mutations known to be associated with cell activation. Next, the authors analyzed the positive and negative sgRNAs for dgkz, LCP2, PIK3CD, PLCG1, and V**1 that were systematically screened to analyze the different functions of different alleles. The authors validated these screened results and found that positive and negative sgRNAs for DGKZ, LCP2, PIK3CD, PLCG1, and V**1 promoted or inhibited cytotoxic function, respectively. The function of the two allele mutations in PIK3CD screened by the authors has been reported to be the immune pathogenic mutations in primary T cells, which further confirms that the system used by the authors can be used to screen for functional alleles. The authors also evaluated the relationship between the screened functional data and the known protein structure. The analysis found that acquired functional mutations in PIK3CD can lead to the 3D pocket structures being concretely put together. and Pik3CD cocrystallized with the PIK3R1 regulatory subunit of PI3K, which was also screened and identified by the authors. Base editing against the PIK3R1 site can increase cellular TNF, IL2, and IFN production. This also suggests that base-editing mutagenesis can provide information about key residues and domains in the protein structure of primary human T cells, including protein-protein interaction sites. The study reveals that base-editing mutagenesis can deepen the understanding of molecular function and facilitate the development of engineered proteins and RNAs, providing a transformative tool for the development of immunity**. Original link:
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