CRISPR/Cas Genome Editing Single Guide RNA (sgRNA) Design using Three Different Web Tool Platforms

Background: Genome editing is a group of technologies that has the ability to change an organism’s DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome. Several approaches to genome editing have been developed. A well-known one is called CRISPR/Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9. Editing through CRISPR focuses mainly on the Protospacer-adjacent motif (PAM), a crucial region to identify the sgRNAs to target the desired gene or region of DNA. So designing precise single-guide RNAs (sgRNAs) to minimize off-target effects is critical for the success of gene editing without undesired results. Methods: Hence, in this article, three different online tools are used to design sgRNA to target the prolactin (PRL) gene to knock out. The Gallus gallus Prolactin (PRL) gene sequence is retrieved from NCBI and used for further downstream application. However, all three web tools may vary in design specifications and parameter choices, visualization, downstream analysis functionality, etc. Result: While keeping a straightforward and interactive interface and running with default parameters, all web tools also accept a variety of advanced choices for more specialized searches. This maximizes user flexibility. Three tools produced several sgRNAs that satisfied various criteria for precise gene editing to boost the efficacy of the target prolactin gene. These online tools use a robust approach to identify off-target locations and the results are displayed in an interactive table and within the gene architecture.