FIGURE 2. Guide RNA and Repair Template Design.

(A) A Plk4 genomic target sequence (underlined) is chosen due to the close proximity of the cut site to the desired mutation (gatekeeper residue L89G, green (gray in print versions)). The PAM site required for SpCas9/gRNA cleavage (red (light gray in print versions)) lies immediately downstream of the genomic target sequence. The 5′ end of the genomic target sequence must start in a G for efficient transcription from the U6 promoter on the PX459 vector. When the genomic target sequence does not begin with a G, this must be added to the gRNA targeting sequence (blue (dark gray in print versions)). To clone the gRNA targeting sequence into the BbsI-digested PX459 vector, the sequence 5′-CACC-3′ must be added onto the 5′ end of the genomic target sequence and the sequence 5′-AAAC-3′ must be added onto the reverse complement of the genomic target sequence. (B) The Plk4^AS repair template contains the gatekeeper residue mutation (L89G), a PAM site mutation, and introduces an AflIII restriction site to facilitate identifying clones that have undergone HDR (all mutations are shown in green (light gray in print versions)). Note that the PAM mutation and restriction enzyme recognition site insertion do not alter the coding sequence.