Molecular Plant Breeding 2024, Vol.15, No.4, 178-186 http://genbreedpublisher.com/index.php/mpb 180 Figure 1 Basic flow chart of the CRISPR/Cas9 genome editing system (Adopted from Fiaz et al., 2019) Image caption: 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) 3.2 Targeted gene editing for yield traits CRISPR/Cas9 technology has been instrumental in the targeted editing of genes to enhance yield traits in rice. By focusing on specific genes that regulate grain size, panicle length, and stress tolerance, researchers have been able to create rice varieties with superior agronomic performance. For example, the simultaneous editing of OsPIN5b, GS3, and OsMYB30 genes led to the development of rice mutants with increased panicle length, enlarged grain size, and enhanced cold tolerance. These modifications resulted in higher yields and better stress resistance, demonstrating the multifaceted benefits of targeted gene editing (Zeng et al., 2020). Furthermore, the editing of the GS3 gene alone has been shown to increase grain length and weight by over 30%, further emphasizing the potential of CRISPR/Cas9 in yield trait improvement (Usman et al., 2021).
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