Clustered regularly interspaced short palindromic repeats (CRISPRs) technique is the most effective and novelist technique for genome editing. CRISPR mechanism has been widely developed for gene editing, gene silencing, high-specific regulation of the transcription, and reducing off-target effects through double-strand breaks (DSBs) in the genomic DNA and then modifying nucleotide sequences of the target gene in diverse plant and animal species. However, the application may be restricted by a high rate of off-target effects. So, there are many studies on designing precise single-guide RNAs (sgRNAs) to minimize off-target effects. Thus, the high-efficiency design of a specific sgRNA is critical. First, in the chapter, the sgRNA origin and different types of gRNA will be outlined. Then, the off-target effect will be described. Next, the remarkable characteristics of the sgRNA will be highlighted to improve precise gene editing. Finally, some popular in silico tools will be introduced for designing sgRNA.