Dayanc Bengisu, Eris Sude, Senturk Serif
Izmir Biomedicine and Genome Center, Izmir, Türkiye.
Izmir International Biomedicine and Genome Institute, Dokuz Eylul University, Izmir, Türkiye.
Bio Protoc. 2025 Feb 20;15(4):e5183. doi: 10.21769/BioProtoc.5183.
Recent advancements in high-throughput functional genomics have substantially enhanced our comprehension of the genetic and molecular dimensions of cancer, facilitating the identification of novel therapeutic targets. One of the key methodological innovations in this field is the CRISPR screening strategy, which has proven efficacy in elucidating essential gene functions and pathway alterations critical to cancer cell survival and fitness. The construction of custom CRISPR libraries permits the integration of tailored single-guide RNAs (gRNAs), offering greater flexibility as well as specificity in comparison to the commercially available libraries, and enables more refined secondary screening strategies to attenuate the selection of false positive potential gene candidates. Among various molecular cloning techniques, circular polymerase extension cloning (CPEC) has emerged as a highly efficient and cost-effective approach. CPEC utilizes polymerase overlap extension to assemble overlapping DNA fragments into circular plasmids, eliminating the need for restriction digestion and ligation and thus streamlining the creation of both single and multi-fragment constructs. In this protocol, we present the application of the CPEC method to construct the EpiTransNuc knockout gRNA library, specifically designed to target epigenetic regulators, transcription factors, and nuclear proteins. The custom library, assembled using the lentiGuide-Puro backbone, comprises 40,820 gRNAs, with 10 gRNAs per gene, along with 100 non-targeting control gRNAs. Importantly, the CPEC method can be tailored to meet the specific requirements of other custom gRNA libraries, offering flexibility for diverse research applications. Key features • Involves PCR-based linearization of the backbone with designed primer sets. • Facilitates flexibility in gRNA composition and number in library construction. • Skips conventional cloning techniques such as restriction digestion and ligation. Graphical overview Schematic representation of the circular polymerase extension cloning (CPEC) procedure.
高通量功能基因组学的最新进展极大地增进了我们对癌症遗传和分子层面的理解，有助于识别新的治疗靶点。该领域关键的方法学创新之一是CRISPR筛选策略，它已被证明在阐明对癌细胞存活和适应性至关重要的必需基因功能及通路改变方面有效。定制CRISPR文库的构建允许整合定制的单向导RNA（gRNA），与市售文库相比，具有更大的灵活性和特异性，并且能够采用更精细的二次筛选策略来减少假阳性潜在基因候选物的选择。在各种分子克隆技术中，环化聚合酶延伸克隆（CPEC）已成为一种高效且经济高效的方法。CPEC利用聚合酶重叠延伸将重叠的DNA片段组装成环状质粒，无需限制性酶切和连接，从而简化了单片段和多片段构建体的创建。在本方案中，我们展示了CPEC方法在构建EpiTransNuc敲除gRNA文库中的应用，该文库专门设计用于靶向表观遗传调节因子、转录因子和核蛋白。使用慢病毒引导嘌呤霉素骨架组装的定制文库包含40,820个gRNA，每个基因有10个gRNA，以及100个非靶向对照gRNA。重要的是，CPEC方法可进行调整以满足其他定制gRNA文库的特定要求，为各种研究应用提供灵活性。关键特性•使用设计的引物组对骨架进行基于PCR的线性化。•在文库构建中便于gRNA组成和数量的灵活性。•跳过诸如限制性酶切和连接等传统克隆技术。图形概述环化聚合酶延伸克隆（CPEC）过程的示意图。
