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

Vector construction and transfection
Human full-length EZH2 (NM_152998.2) cDNA was amplified by reverse transcription polymerase chain reaction using mRNA extracted from HeLa cells. The primer sequences were designed as follows:

EZH2-F: 5-′ CCGCTCGAGGCCACCATGGGCCAGACTGGGAAGAAATCTG-3′; and EZH2-R: 5-′ CGCGGATCCTCAAGGGATTTCCATTTCTCTTTCG-3′

The EZH2 DNA fragment was subsequently cloned into the Xho I and BamH I (TaKaRa, Tokyo, Japan) sites of an internal ribosome entry site vector, pIRES2-AcGFP1 (Clontech, Mountain View, CA), to generate the pIRES2-AcGFP1-EZH2 recombinant plasmid.

The pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Plasmid#42230) containing SpCas9 and single guide RNA was obtained from a non-profit plasmid share repository belonging to Feng Zhang (Addgene, Cambridge, MA, USA) [64]. Suitable CRISPR target sites within the positive and negative strands of exon 2 of EZH2 were identified using the ‘CRISPR Design Tool' (http://crispr.mit.edu/) hosted by the Feng Zhang laboratory (Massachusetts Institute of Technology, MA, USA) to obtain the minimum number of off-target sites in the human genome (Supplementary Figure 1A). The respective oligonucleotide pairs were synthetized by GenScript Technologies (Nanjing, China) and were customized to include overhangs compatible for ligation into Plasmid#42230 linearized by digestion with BbsI (New England BioLabs, Ipswich, MA, USA), and these overhangs were located in two places in this vector carrying a guide sequence. Oligonucleotides were annealed and inserted into the plasmid by DNA ligation (TaKaRa, Tokyo, Japan). Then, to prevent screening issues, we structured an additional homologous recombinant plasmid that included the screening marker G418. The two plasmids were co-transfected. Positive clones were verified by Western blot analysis and DNA sequencing of genomic PCR products encompassing the CRISPR target sites (Supplementary Figure 1B and Figure 2A1).

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Discussion

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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