CRISPR Guide RNA Design and Delivery

CRISPR target sites were selected based on their proximity to the start and stop codons of the coding sequence of the targeted gene, and also by the requirement for a protospacer adjacent motif (PAM) sequence (NGG) at the 3′ end of target site. We created and annealed oligos containing a T7 promoter sequence, the target sequence, and an additional constant region to create the template for the guide RNAs (Additional file 1). These templates were transcribed in vitro using T7 RNA polymerase (Promega) in a reaction containing transcription buffer (Promega), RNase inhibitor (Promega), and rNTPs. A linearized plasmid encoding cas9 [26 (link)] was also transcribed in vitro using the Sp6 mMessage mMachine Kit (Ambion). The two guide RNAs targeting each gene were combined with cas9 mRNA and phenol red, and 1-2 nl of this mixture was injected into the cell of early 1-cell stage embryos.