pX330-U6-Chimeric_BB-CBh-hSpCas9

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

Plasmids for the multiplex CRISPR/Cas9 vector system
All plasmids for the multiplex CRISPR/Cas9 vector system were constructed using the In-Fusion cloning method with PCR products from pX330 (Addgene, Cambridge, MA; Plasmid 42230)4,8 and pFUS_A30A (Addgene; Plasmid 31029)27 as summarized in Supplementary Table S1.

Insertion of annealed oligonucleotides into pX330A and pX330S vectors
To construct CRISPR/Cas9 plasmids targeting human genes, sense and antisense oligonucleotides were synthesized and annealed in the following buffer: 40 mM Tris-HCl (pH 8.0), 20 mM MgCl2, and 50 mM NaCl. The annealed oligonucleotides, pX330A/S vectors, BpiI enzyme (Thermo Scientific, Rockford, IL), and Quick ligase (New England Biolabs, Beverly, MA) were mixed in a single tube with T4 DNA ligase buffer (New England Biolabs), and subjected to a thermal cycling reaction as follows: 3 cycles of 37°C for 5 min and 16°C for 10 min. After the cycling reaction, additional BpiI digestion was performed at 37°C for 1 h. A list of the constructed plasmids with the vectors used and oligonucleotide sequences is shown in Supplementary Table S2.

Golden Gate assembly and screening by colony PCR
Golden Gate assembly was performed as described previously27,28,29 with some modifications. pX330A/S plasmids, BsaI-HF enzyme (New England Biolabs), and Quick ligase were mixed in a single tube with T4 DNA ligase buffer, and subjected to a thermal cycling reaction as follows: 6–15 cycles of 37°C for 5 min and 16°C for 10 min. After the cycling reaction, additional BsaI-HF digestion was performed at 50°C for 30 min. For construction of the CRISPR/Cas9-nuclease vector targeting seven genomic loci, pX330A-1x7_HPRT1_A, pX330S-2_HPRT1_B, pX330S-3_ATM, pX330S-4_APC, pX330S-5_CDH1, pX330S-6_AXIN2, and pX330S-7_CFTR were unified into a single vector. For construction of the CRISPR/Cas9-nickase vector targeting three genomic loci, pX330A_D10A-1x6_off-5_L, pX330S-2_off-5_R, pX330S-3_off-8_L, pX330S-4_off-8_R, pX330S-5_off-4_L, and pX330S-6_off-4_R were unified into a single vector.

Correctly-assembled clones were screened by colony PCR using CRISPR-step2-F (5′-GCCTTTTGCTGGCCTTTTGCTC-3′) and CRISPR-step2-R (5′-CGGGCCATTTACCGTAAGTTATGTAACG-3′) primers, followed by agarose gel electrophoresis and ethidium bromide staining.



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Manufacturer protocol

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

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