dsDAP: an efficient method for high-abundance DNA-encoded library construction in mammalian cells

DNA-encoded libraries are invaluable tools for high-throughput screening and functional genomics studies. However, constructing high-abundance libraries in mammalian cells remains challenging. Here, we present dsDNA-assembly-PCR (dsDAP), a novel Gibson-assembly-PCR strategy for creating DNA-encoded libraries, offering improved flexibility and efficiency over previous methods. We demonstrated this approach by investigating the impact of translation initiation sequences (TIS) on protein expression in HEK293T cells. Both CRISPR-Cas9 and piggyBac systems were employed for genomic integration, allowing comparison of different integration methods. Our results confirmed the importance of specific nucleotides in the TIS region, particularly the preference for adenine at the -3 position in high-expression sequences. We also explored the effects of library dilution on genotype-phenotype correlations. This Gibson-assembly-PCR strategy overcomes limitations of existing methods, such as restriction enzyme dependencies, and provides a versatile tool for constructing high-abundance libraries in mammalian cells. Our approach has broad applications in functional genomics, drug discovery, and the study of gene regulation. Overall design: We developed a novel DNA-encoded library construction strategy named dsDNA-assembly-PCR (dsDAP). This dsDNA-based method utilizes Gibson assembly for library construction, eliminating the need for restriction endonucleases. As a result, the library preparation process becomes more straightforward and feasible compared to the in-vitro-ligation-PCR strategy. By stably integrating the dsDAP library into the HEK293T cell genome through the CRISPR-Cas9 and piggyBac systems, we investigated the impact of translation initiation sequences (TISs) on translation efficiency.

DNA编码文库（DNA-encoded libraries）是高通量筛选与功能基因组学研究中极具价值的工具。然而，在哺乳动物细胞中构建高丰度文库仍存在诸多挑战。本研究提出一种新型Gibson组装PCR（Gibson-assembly-PCR）策略——双链DNA组装PCR（dsDNA-assembly-PCR，简称dsDAP），用于制备DNA编码文库，相较于既往方法具备更优的灵活性与效率。
我们通过探究翻译起始序列（translation initiation sequences，简称TIS）对HEK293T细胞内蛋白质表达的影响，验证了该方法的有效性。实验中同时采用CRISPR-Cas9与piggyBac系统完成基因组整合，得以对比不同整合方式的效果。研究结果证实了TIS区域特定核苷酸的重要性，尤其在高表达序列中，-3位核苷酸偏好腺嘌呤。我们还探讨了文库稀释对基因型-表型相关性的影响。
该Gibson组装PCR策略克服了现有方法依赖限制性内切酶等局限，为哺乳动物细胞内高丰度文库的构建提供了一款通用性极强的工具。本方法在功能基因组学、药物发现以及基因调控研究中拥有广泛的应用场景。
整体设计：我们开发了一种名为双链DNA组装PCR（dsDNA-assembly-PCR，简称dsDAP）的新型DNA编码文库构建策略。该基于双链DNA的方法利用Gibson组装完成文库构建，无需使用限制性内切酶，相较于体外连接PCR（in-vitro-ligation-PCR）策略，文库制备流程更为简便可行。我们通过CRISPR-Cas9与piggyBac系统将dsDAP文库稳定整合至HEK293T细胞基因组中，以此探究翻译起始序列（TIS）对翻译效率的影响。