CRISPRi-FGP: web-based genome-scale CRISPRi sgRNA design and validation tool in prokaryotes

CRISPRi screening has become a powerful approach for functional genomic research. However, the off-target effects resulting from the mismatch tolerance between sgRNAs and their intended targets is a primary concern in CRISPRi applications. To address this issue, we introduce Guide Library Designer (GLiDe), a web-based tool specifically created for the genome-scale design of sgRNA libraries tailored for CRISPRi screening in prokaryotic organisms. GLiDe incorporates a robust quality control framework, rooted in prior experimental knowledge, ensuring the accurate identification of off-target hits. It boasts an extensive built-in database, encompassing 1,397 common prokaryotic species as a comprehensive design resource. In addition, GLiDe provides the capability to design sgRNAs for newly discovered organisms. We further demonstrated that GLiDe exhibits enhanced precision in identifying off-target binding sites for the CRISPRi system. Availability and ImplementationFreely available on the web at: https://www.thu-big.net/sgRNA_design/. AUTHOR SUMMARYCRISPRi screening is a powerful strategy for functional genomic study in prokaryotes. However, its applicability has been hindered by false positive results arising from off-target binding of sgRNAs. To tackle this challenge head-on, we present GLiDe, a web-based tool meticulously crafted for the genome-scale CRISPRi sgRNA library design in prokaryotes. GLiDe integrates a stringent quality control mechanism grounded in prior experimental insights. It has a built-in database and accepts uploaded reference files for new organisms. Moreover, we have demonstrated that our tool outperforms existing tools in CRISPRi sgRNA design for prokaryotes through cytometry assay. In summary, GLiDe emerges as a robust solution for sgRNA library design, poised to significantly advance functional genomic studies.