Abstract Background 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. Results We introduce Guide Library Designer (GLiDe). https://www.roneverhart.com/Fleshripper-Ethereal-275-ED-East-Non-Ladder/