Pcag t3 hcas pa

We constructed the plasmid vector px330 (Addgene #42230) expressing single-guide RNA (sgRNA) as well as Cas9. The px330 plasmid^{9 (link)} was a gift from Feng Zhang, and the sgRNA was designed in silico via the CRISPR Design tool^{29 (link)} or sgRNA designer^{30 (link)}. For in vitro synthesis of Cas9 mRNA, plasmid vector pCAG-T3-hCAS-pA (Addgene #48625) was linearized by SphI, then transcribed with T3 RNA polymerase (Promega, Madison, WI, USA) in the presence of Ribo m⁷G Cap Analog (Promega) as described previously^{31 (link)}. pCAG-T3-hCAS-pA was a gift from Wataru Fujii & Kunihiko Naito. sgRNAs were synthesized in vitro as previously described^{32 (link)} using a MEGAshortscript T7 kit (Thermo Fisher Scientific, Waltham, MA, USA). Synthesized RNAs were purified using a MEGAclear kit (Thermo Fisher Scientific). All sequences of oligonucleotides for sgRNA synthesis are listed in Supplementary Table 1.

For synthesis of Cas9 mRNA in vitro, plasmid vector pCAG-T3-hCAS-pA (Addgene 48625) was linearized by Sph I, then transcribed with T3 RNA polymerase (Promega) in the presence of Ribo m⁷G Cap Analog (promega) as previously described [31 (link)]. The MEGAshortscript T7 (Thermo Fisher Scientific AM1354) and MEGAclear (Thermo Fisher Scientific AM1908) kits were used for in vitro transcription of sgRNAs, while the CRISPR Design tool was used for creating sgRNAs [32 (link)]. All oligonucleotide sequences used for in vitro transcription are listed in Supplementary Table S4.

For the in vitro synthesis of gRNAs, the T7 promoter was inserted upstream of gRNAs by PCR using oligomers as previously described31 (link), and 5′-GG transcription start region was arbitrarily created (Supplementary Table S4). The T7-gRNAs were gel-purified for in vitro transcription. Both std-gRNAs and tru-gRNAs were transcribed in vitro with the MEGAshortscript Kit (AM1354, Ambion). The pCAG-T3-hCAS-pA (#48625, Addgene) plasmid was digested by NruI, and the DNA fragment encoding Cas9 was recovered by gel extraction for in vitro transcription. Cas9 mRNA was transcribed with the mMESSAGE mMACHINE^® T3 Transcription Kit (AM1348, Ambion). All RNAs were purified with the MEGAclear Transcription Clean-Up Kit (AM1908, Ambion) and stored at −80° C until use.