Nucleotides 51–53 of the tracrRNA should be not paired to the seed region and be freely accessible, and there should be favorable nucleotides at several positions 17 Once the PAM is recognized, the guide region of the gRNA undergoes seed nucleation to form an Aformlike helical RNADNA hybrid duplex The PS2– and PS3–gRNA seeds region ( and 22 nt, respectively) were predicted to pair with the coding strand of OsMPK5, and PS3–gRNA would guide Cas9 to make DSB at a SacI site Subsequently, three gRNA–Cas9 constructs were made by inserting the synthetic DNA oligonucleotides which encode the gRNA seed into the pRG vector
Crispr Cas3 Induces Broad And Unidirectional Genome Editing In Human Cells Nature Communications
