Construction of an expression plasmid for sgRNA is also simple and rapid, involving a single cloning step with a pair of partially complementary oligonucleotides. The oligo pairs encoding the 20-nt guide sequences are annealed and ligated into a plasmid (pSpCas9(BB), Fig. 4c) bearing both Cas9 and the remainder of the sgRNA as an invariant scaffold immediately following the oligo cloning site. The transfection plasmids can also be modified to enable virus production for in vivo delivery. For these approaches, the following plasmids are used within this protocol: Cas9 alone (pSpCas9) or Cas9 with an invariant sgRNA scaffold and cloning sites for inserting a guide sequence (pSpCas9(BB)). For the backbone cloning construct, we have also fused 2A-GFP or 2A-Puro to Cas9 to allow screening or selection of transfected cells (pSpCas9(BB)-2A-GFP or pSpCas9(BB)-2A-Puro, respectively). Finally, we provide pSpCas9n(BB), a D10A nickase mutant of Cas9 for HDR and for double-nicking applications (Box 2), along with the 2A-GFP and 2A-Puro fusion constructs (pSpCas9n(BB)-2A-GFP, pSpCas9n(BB)-2A-Puro).
