Paav nefcas9

To facilitate the generation of knock-in constructs, we developed a simple template vector (pORANGE). For this, we used pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene 62988) and replaced SpCas9puro by SpCas9 from pAAV-nEFCas9 (Addgene 87115) flanked by the bipartite SV40 nuclear localization signal (NLS) sequences using the AgeI and EcoRI restriction sites, generating pSpCas9. To facilitate cloning of donor sequences, a multiple cloning site was inserted by annealing two complementary DNA oligos and ligation into the XbaI site of pSpCas9 generating pORANGE.

The gRNA cloning plasmid (gRNA_cloning vector), GFP-expression self-complementary AAV plasmid (pscAAV-CAG-GFP), GFP-containing plasmid (pCAG-1BPNLS-Cas9-1BPNLS-2AGFP), Cas9-expression AAV plasmid (pAAV-nEFCas9), and AAV donor backbone plasmid (pAAV-rMERTK-HITI) were obtained from Addgene (Addgene 41824, 83279, 87109, 87115, and 87119, respectively). To construct the myosin heavy chain 6 (Myh6) target gRNA-expression vector (mMyh6-gRNA), we designed the Myh6 target sequence (20 bp target and 3 bp PAM sequence (underlined)) as follows: GCAGCAGAAGATGCACGACGAGG. The Myh6 target was subcloned into the gRNA_cloning vector according to a previously reported protocol (https://media.addgene.org/data/93/40/adf4a4fe-5e77–11e2–9c30–003048dd6500.pdf (20 April 2020, date last accessed)) with the primers 5′- TGGCTTTATATATCTT
GTGGAAAGGACGAAACACCGCAGCAGAAGATGCACGACG-3′ and 5′- GCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCGTCGTGCATCTTCTGCTGC-3′. To construct a donor/gRNA AAV backbone plasmid (pAAV-Myh6GFP-HITI) for GFP knock-in at the Myh6 locus, U6-mMyh6gRNA and mMyh6GFP-HITI fragments were amplified from mMyh6-gRNA and pCAG-1BPNLS-Cas9-1BPNLS-2AGFP, respectively, using PrimeSTAR GXL DNA polymerase (Takara Bio, Inc., Shiga, Japan). These PCR fragments were subcloned into pAAV-rMERTK-HITI using an In-fusion HD Cloning Kit (Takara Bio, Inc.).