AbstractCapitalizing on the inherent multiplexing capability of AsCpf1, we developed a multiplexed, high-throughput screening strategy that minimizes library size without sacrificing gene targeting efficiency. We demonstrated that AsCpf1 can be used for functional genomics screenings and that an AsCpf1-based multiplexed library performs similarly as compared to currently available monocistronic CRISPR/Cas9 libraries, with only one vector required for each gene. We construct the smallest whole-genome CRISPR knock-out library, Mini-human, for the human genome (n = 17,032 constructs targeting 16,977 protein-coding genes), which performs favorably compared to conventional Cas9 libraries.