ZFIN Image: Yates et al., 2021, Fig. 2

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Figure Caption

Fig. 2

Incorporation of the MmeI Recognition Sequence into the Repeat:Anti-Repeat Duplex and Elution with a Strand Displacing Polymerase. (A) Modifications were made to the Repeat:Anti-Repeat Duplex of the sgRNA sequence to insert a single MmeI site that cleaves 18-20 bp upstream of the sgRNA recognition sequence. Modifications are shown in red. The MmeI binding sites are shown in dark gray boxes. The MmeI cut site is represented with a triangle. (B) in vitro Cas9 digestion of a 1000 bp DNA fragment with each sgRNA variant. (C) Injection of zebrafish embryos with Cas9 and the sgRNA variant targeting the gol gene compared to an uninjected control (minimum n per pool = 150). (D) Detachment of the sgRNA scaffold from magnetic beads using Bst 3.0 or Pol1.

Acknowledgments

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