pX330-U6-Chimeric_BB-CBh-hSpCas9

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Publication protocol

Cloning the CRISPR Construct
The hSpCas9 nuclease-expressing pX330 plasmid (no. 42230)30 was purchased from Addgene. Using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), we modified the sgRNA cloning sites of pX330 by changing the BbsI recognition site (GAAGAC) to that of BsaI (GGTCTC). To generate U6-sgRNA-Cas9-IRES-mCherry plasmids, we PCR amplified IRES-mCherry with IRES3-mCherry-CL plasmids (kindly provided by Prof. Chang Hwan Park of Hanyang University) as a template and inserted it between the Cas9 sequence and the 3′ nuclear localization sequence of pX330. To screen sgRNAs and generate knockout clones, we used a plasmid encoding Cas9-2A-mRFP-2A-PAC (puromycin N-acetyl-transferase, puromycin resistance gene) and a plasmid encoding sgRNAs (both from ToolGen). The sgRNA target sequences were manually designed on the basis of the protospacer adjacent motif (PAM) sequence (NGG) and cloned into the vectors as previously described.31 In brief, oligonucleotides containing each target sequence were synthesized (Bioneer) and annealed in vitro with a thermocycler. The vector was digested with BsaI and ligated with the annealed oligonucleotides. Oligonucleotide sequences are listed in Table S7.

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Papers

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Manufacturer protocol

Download the product protocol from Addgene for pX330-U6-Chimeric_BB-CBh-hSpCas9 below.

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Videos

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