pSpCas9(BB)-2A-Puro (PX459)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Protocol tips
There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

Construction of tru-RGN and std-RGN expression plasmids
Target sites at the second exon of the FVII gene (Supplementary Fig. S1) were analyzed by the MIT CRISPR Design Tool22. Off-target sites were identified in parallel, and gRNAs with the highest scores were chosen. Tru-gRNAs contained 17 or 18 target complementary nucleotides shortened from std-gRNAs. Oligomers encoding gRNAs were synthesized (Supplementary Table S4), annealed, and cloned into the PX459 vector (#48139, Addgene) at its BbsI site as previously described22. An artificial BbsI restriction site was generated for each vector by adding 4–5 nucleotides (Supplementary Table S3). Recombinant plasmids were sequenced and subsequently used for cell transfection.

gRNA and Cas9 mRNA in vitro transcription
For the in vitro synthesis of gRNAs, the T7 promoter was inserted upstream of gRNAs by PCR using oligomers as previously described31, 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.

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Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-Puro (PX459) below.

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