pSpCas9(BB)-2A-GFP (PX458)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

2.2. Construction and Validation of Customized CRISPR/CAS9 Expression Vectors
The vector pSpCas9(BB)-2A-GFP (PX458) (Plasmid #48138) was purchased from Addgene (Massachusetts, USA). The oligo-DNA targeting the PAX5 exon5 locus was designed on the MIT online software ZhangFeng lab: http://crispr.mit.edu/. We selected three high scored sequences and designed their respective complement chains with restriction site; each single strand oligo-DNA chain was synthesized in Invitrogen company as follows:

  PAX5 gRNA-F1: cacc  GACAAAAGTACAGCAGCCAC

  PAX5 gRNA-R1: aaac  GTGGCTGCTGTACTTTTGTC

  PAX5 gRNA-F2: cacc  AACCAACCAGTCCCAGCTTC

  PAX5 gRNA-R2: aaac  GAAGCTGGGACTGGTTGGTT

  PAX5 gRNA-F3: cacc  ACCAACCAGTCCCAGCTTCC

  PAX5 gRNA-R3: aaac  GGAAGCTGGGACTGGTTGGT

Next, we annealed the two complement chains to form dsDNA using Precut sgRNA Cloning kit and pSD-gRNA Plasmid construction Kit (Biomics Biotechnologies Co., Ltd., Jiangsu, China) according to the instruction. This was followed by BbsI digestion and ligation with T4 ligase to construct Cas9/sgRNA plasmids targeting PAX5. The Cas9/sgRNA plasmids were amplified, purified with EndoFree Plasmid Maxi Kit (QIAGEN, Germany), and validated by sequencing. The Cas9/sgRNA plasmids were electrotransfected into HEK293T cells. The T7 Endonuclease I assay was applied to measure the NHEJ-mediated mutations efficiency in the endogenous PAX5 gene. The most efficient Cas9/sgRNA plasmid (gRNA-F1/R1) was chosen for subsequent research.



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Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-GFP (PX458) below.

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