Abstract
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.