Functional genetic screening using the CRISPR/Cas9 system is a powerful tool for interrogating gene function at scale, but access to and implementation of this technology is constrained by existing pre-defined CRISPR sgRNA libraries. Here, a single Twist Oligo Pool was used in combination with Custom Library Multiplexed Cloning (CLUE; crispr-clue.de), an easy-to-use bioinformatics and wet-lab workflow, to generate numerous high-quality, custom sgRNA libraries. The exceptional fidelity of Twist Oligo Pools preserves the uniform representation of sgRNAs in the resulting CLUE libraries without cross-library contamination, meeting the stringent requirements for success in CRISPR screening applications.