CRISPR-based screening has emerged as a powerful tool for identifying new gene targets for desired cellular phenotypes. The construction of gRNA pools largely determines library quality and is usually performed using Golden Gate assembly, or Gibson assembly. To date, library construction methods have not been systematically compared, and the quality check of each batch has been slow. In this study, an in-house nanopore sequencing workflow was established for assessing current methods of gRNA pool construction. The bias of pool construction was reduced by employing the polymerase-mediated non-amplifying method. Then, a small gRNA pool was utilized to characterize stronger activation domains, specifically MED2 (a subunit of mediator complex) and HAP4 (a heme activator protein), as well as to identify better gRNA choices for dCas12a-based gene activation in S. cerevisiae. Furthermore, based on the better CRISPRa tool identified in this study, a custom gRNA pool, which consisted of 99 gRNAs targeting central metabolic pathways, was designed and employed to screen for gene targets that could improve ethanol utilization in S. cerevisiae. The nanopore sequencing based workflow demonstrated here should provide a cost-effective approach for assessing quality of customized gRNA library, leading to faster and more efficient genetic and metabolic engineering in S. cerevisiae.