Rice Genomics and Genetics 2025, Vol.16, No.4, 237-244 http://cropscipublisher.com/index.php/rgg 239 Figure 1 Basic flow chart of the CRISPR/Cas9 genome editing system. The engineered CRISPR/Cas9 system consist of two components; (1a) the Cas9 endonuclease and, (1b) a single-guide RNA (sgRNA). “The sgRNA contains a spacer sequence followed by 79 nt of an artificially fused tracrRNA and crRNA sequence”, (2) The spacer sequence is typically 20 nt in length, and specifically binds to the target DNA sequence containing a 5’-NGG-3’ PAM motif at the 3’ end, which is highly specific for the gene of interest, (3) The fused trans-activating crRNA (tracrRNA) and crRNA sequence forms a stem-loop RNA structure that binds to the Cas9 enzyme; tracrRNA hybridizes and joins Cas9. (4) Assembly of sgRNA, attached with the target sequence and the Cas9 vector construct. (5) Transformation of the vector construct into rice via different transformation techniques. (5a) Screening and selection of rice mutant plants based on phenotypic changes. (5b) Restriction enzyme site loss generating a CRISPR/Cas9 mutagenized plant line. (c, control; m, mutagenized; RE, restrictions enzyme). (5c) Surveyor Assay (CEL1 and T7 are DNA endonucleases utilized in surveyor assay). (5d) Next-generation sequencing. (6) Future analysis to obtain T-DNA-free plants, and further experiments to prove phenotypic changes cast by the knockout of the gene under investigation. * Different techniques for the vector construct transformation. ** Regeneration and screening of transgenic plants for gene editing events (Adopted from Fiaz et al., 2019)
RkJQdWJsaXNoZXIy MjQ4ODYzNA==