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Background and Aim: Genome editing is an efficient and accurate method in biological and medical studies. Among the wide range of genome editing techniques, clustered regularly interspaced short palindromic repeats (CRISPR) is one of the simplest and yet promising methods. The CRISPR system consists of two key components; an endonuclease enzyme called Cas9 and guide RNA (gRNA), ensuring that Cas9 enzyme cuts at the right point in the genome. Although CRISPR genome editing is one of the useful methods to modify the genome, there are still multiple challenges in the technique steps. Materials and Methods: sgRNAs were designed and ordered accordingly on the basis of the target site and vector of interest. Cloning procedures were performed and confirmed by sequencing as a gold standard. The list of high efficient sgRNAs for the LAMP-2 gene was prepared based on the available outstanding databases by analyzing and comparing the specificity and functionality as two main characteristics. Results: In this study, we tried to evaluate the main challenges in the process of preparing CRISPR/Cas9 popular vectors (pX330/pX459). A list of high efficient sgRNAs for an autophagy gene is also prepared as an example for clarification of the sgRNA design procedures. Conclusion: This study tried to depict problems that may be encountered during sgRNA design and plasmid preparation, followed by giving appropriate recommendations.