Full gRNAmRNA hybridization is triggered once the gRNA critical seed region (nt2nt4) binds to the target mRNA The fully hybridized structure is herein termed the activatedRISC 16 The target We designed seven gRNAs targeting a region of EPSPS that contains the universal mutation hotspot for resistance to glyphosate (Fig 1) gRNA1, gRN and gRN were designed to target only one or two of the three EPSPS homoeoalleles, whereas the other four gRNAs were designed to target all three homoeoallelesSilent protospacer adjacent motif (PAM) site mutations (Gaa c11C>A, Gaa c1845G>A) and a gRNA seed region mutation (Gaa c1842A>T) were also introduced to prevent gRNA editing of the donor template sgRNA and the Cas9 nuclease were introduced into the cytoplasm of C57BL/6NJderived zygotes with well recognized pronuclei Correctly targeted embryos were transferred to
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