Tree Genetics and Molecular Breeding 2024, Vol.14, No.1, 32-42 http://genbreedpublisher.com/index.php/tgmb 35 Figure 2 RNA-guided DNA cleavage by Cas9 (Adopted from Bortesi and Fischer, 2015) Image caption: (a) In the native system, the Cas9 protein (light blue) is guided by a structure formed by a CRISPR RNA (crRNA, in black), which contains a 20-nt segment determining target specificity, and a trans-activating CRISPR RNA (tracrRNA, in red), which stabilizes the structure and activates Cas9 to cleave the target DNA (protospacer). The presence of a protospacer-adjacent motif (PAM, in yellow), i.e., an NGG (or less frequently NAG) sequence directly downstream from the target DNA, is a prerequisite for DNA cleavage by Cas9. Among the 20 RNA nucleotides determining target specificity, the so-called seed sequence of approximately 12 nt (in orange) upstream of the PAM is thought to be particularly important for the pairing between RNA and target DNA. (b) Cas9 can be reprogrammed to cleave DNA by a single guide RNA molecule (gRNA, in green), a chimera generated by fusing the 3’ end of the crRNA to the 5’ end of the tracrRNA (Adopted from Bortesi and Fischer, 2015) Figure 3 Genome editing with site-specific nucleases (Adopted from Bortesi and Fischer, 2015) Image caption: Double-strand breaks induced by a nuclease at a specific site can be repaired either by non-homologous end joining (NHEJ) or homologous recombination (HR). (a) Repair by NHEJ usually results in the insertion (green) or deletion (red) of random base pairs, causing gene knockout by disruption. (b) If a donor DNA is available, which is simultaneously cut by the same nuclease leaving compatible overhangs, gene insertion by NHEJ can also be achieved. (c) HR with a donor DNA template can be exploited to modify a gene by introducing precise nucleotide substitutions or (d) to achieve gene insertion (Adopted from Bortesi and Fischer, 2015)
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