Maize Genomics and Genetics 2024, Vol.15, No.4, 204-217 http://cropscipublisher.com/index.php/mgg 208 Figure 2 CRISPR-based adaptive immunity provides programmable genome editing tools (Adopted from Wang and Doudna, 2023) Image caption: (A) CRISPR immune systems target DNA or RNA in microbes (illustration depicts DNA targeting). Three steps to immunity include: (i) acquisition of CRISPR spacer sequence matching an infectious agent; (ii) transcription and formation of Cas-RNA complexes; (iii) seek-and-destroy surveillance mechanisms. (B) CRISPR-Cas9 is the canonical genome editing tool for RNA-guided genetic manipulation. Cas9 searches for target sites in a genome by engaging with PAM sequences, forming an R-loop with complementary DNA, generating a double-strand DNA (dsDNA) break, and finally releasing DNA for repair (Adopted from Wang and Doudna, 2023) CRISPR technology functions by utilizing a guide RNA (gRNA) to direct the Cas9 nuclease to a specific DNA sequence, where it introduces a double-strand break. This break can then be repaired by the cell's natural repair mechanisms, either through non-homologous end joining (NHEJ) or homology-directed repair (HDR), leading to targeted genetic modifications (Ran et al., 2013; Bortesi and Fischer, 2015). Recent advancements have expanded the CRISPR toolbox to include base editors, prime editors, and CRISPR interference (CRISPRi) and activation (CRISPRa) systems, which allow for more precise and varied genetic modifications (Kumlehn et al., 2018; Monsur et al., 2020; Li et al., 2021). 4.2 Applications of CRISPR in maize trait improvement CRISPR technology has been instrumental in advancing maize breeding by enabling the precise modification of genes associated with important agronomic traits. Researchers have successfully used CRISPR/Cas9 to enhance traits such as yield, stress tolerance, and nutritional content in maize (Kumlehn et al., 2018; Li et al., 2021; Wang and Doudna, 2023). One notable application is the improvement of maize's resistance to biotic and abiotic stresses. By targeting specific genes involved in stress response pathways, scientists have developed maize varieties with enhanced tolerance to drought, pests, and diseases. For example, CRISPR/Cas9 has been used to knock out genes that negatively regulate stress responses, thereby increasing the plant's resilience (Kumlehn et al., 2018; Li et al., 2021).
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