CRISPR/Cas is a natural part of the adaptive immune system in prokaryotes (bacteria and archaea), which protects them from invading nucleic acids such as viruses. The translation of the CRISPR/Cas system from immunity to genome editing tool took place when it was shown that editing could be reprogrammed by changing only 20 bp in the single guide RNA (sgRNA). The sgRNA is a chimera of the naturally occurring CRISPR RNA (crRNA) that is complementary to the target DNA sequence, and the trans-encoded CRISPR RNA (tracrRNA), which forms a structural bridge (scaffold) between the crRNA and Cas nuclease. These two RNA molecules (crRNA and tracrRNA) can be fused artificially to form a chimaeric RNA molecule termed as sgRNA. The sgRNA spacer region can then anneal to a complementary DNA sequence (protospacer) with Cas, and create a double-strand break (DSB) in the gene sequence. The most commonly used Cas protein, Cas9 endonuclease is derived from Streptococous pyogenes and requires conserved NGG (N, any nucleotide; G, guanine) PAM sequence for DNA targeting. PAM is known as a protospacer adjacent motif (PAM) located immediate downstream of the target DNA strand. The specificity is provided by the so-called ‘seed sequence’ approximately 12 bases upstream of the PAM, which must match between the sgRNA spacer and target DNA. However, imperfectly matched spacer sequences can result in cleavage at off-target positions, which means that sgRNA target sequence must be chosen carefully to avoid off-target sites in the genome. The software-based in-silico analysis of available genome sequences from representative plant species can be used to predict specific sgRNA spacers with no or minimal off-target effect. Several bioinformatic tools such as CrisprGE, GenomeCRISPR, CHOPCHOP, SSFinder, CRISPR-P, Cas-OFFinder and Breaking-Cas can be used for precise and off-target free sgRNA designing. The genome-edited lines can be confirmed by DNA sequencing and T7 endonuclease method. This presentation will also be covered the practical application of sgRNA designing and editing confirmation approaches used in our banana biofortification programme.