NGS for library QC and analysis of phenotypic screening. sgRNA library generation and phenotypic screening

High-throughput phenotypic screening based on CRISPRi is a useful tool to investigate the genotypes that make the phenotypes of interest. However, the rational design and synthesis of sgRNA library is time consuming, expensive and even impossible for many organisms because of insufficient genetic information. In this study, a random sgRNA library generation method via enzymatic reactions applicable to any organisms including prokaryotes was developed and utilized to phenotypic screening of E. coli K-12 MG1655 as a proof-of-concepts. Using the total RNA of MG1655, about 90,000-membered sgRNA library was generated. Sequencing result revealed that the generated sequence satisfied the requirement of sgRNA such as spacer length, PAM, and non-template strand selectivity which is necessary for CRISPRi. The generated sgRNA library was used to screening of MG1655 gene essentiality, showing high agreement with the reference. This library generation method provides a powerful tool for genotype-phenotype mapping of numerous organisms with untouched, useful characteristics, but without high-quality genome information.