CRISPR-Cas9 has proved to be an efficient method for genome editing across many living organisms including plants. It is quicker, precise, cheaper, and relatively less complicated technique and has been widely used to induce genetic modifications in plants. Leaf shape is one of the most important phenotypic traits for plant identification and classification. In many crops and ornamentals, leaf shape holds an integral economic value as well. This study was conducted to alter the leaf shape in Petunia by developing an optimized CRISPR-Cas9 system to efficiently knock-out gene(s) responsible for the leaf shape. Knotted-1 gene controls leaf shape by cell division and elongation and it is present in Arabidopsis, Tomato, Tobacco and in Petunia as well. Experiment was performed at Plant in-vitro biotechnology lab, Ghent University, to regenerate petunia plants from available hybrid petunia cultured plants. Gene knock-out studies were also done for the genes responsible for the leaf shape in Solanaceae family. Amplification primers were designed, and PCR protocol was developed and optimized. To design the oligos for CRISPR-Cas9, sequence of the gene was required. Several primers were designed to amplify fragments of different lengths for gene sequencing. As the plant was hybrid, PCR cloning procedure using agrobacterium has been followed to sequence both alleles of the Kn1 gene separately. Results showed that shoot regeneration of Petunia by stem cuttings in MS media has been very efficient providing an overall 95% success rate while the regeneration through leaf culture as well as agrobacterium co-culture has not been successful providing varying results in different experiments. An overall 90% of leaves showed callus growth but 0% shoot growth and 0% Agrobacterium transformation GFP expression was observed. The gene sequencing showed that both alleles of the gene had many single nucleic polymorphic sites (SNPs) which made it difficult to select a specific gRNA with a suitable restriction enzyme site, for agrobacterium expression by leaf infiltration. More studies should be conducted on CRISPR-Cas gene knock-out in inbred lines and further experimentation is required to optimize heterozygous gene sequencing and to establish an accurate CRISPR-Cas9 construct.