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

Cas9/ sgRNA efficiency test in cells
The sgRNAs were designed online as shown in Table ​Table11 and cloned into Px330 or a lentiviral plasmid. The transfection procedure was carried out using lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. Briefly, the cells were transfected with Px330 containing the sgWisp2 plasmid or basic plasmid by lipofectamine 2000 in a 6-well culture plate. Then, 48 hours after the transfection, genomic DNA was extracted from the cells with a lysis buffer. Next, the DNA was precipitated with alcohol and sodium acetate. Subsequently, a T7EN1 (NEB, New England Biolabs, USA) cleavage assay was performed as described by Shen et al [44]. Briefly, the targeted fragments from the genomic DNA were amplified by a 2×Taq PCR Mix (Ald lab, Beijing, China) and purified using a TYIAN Quick Midi Purification Kit (Tiangen, Beijing, China). The primers used to amplify the Wisp2 targeted fragments are listed in Table ​Table2.2. The purified PCR products were denatured and re-annealed in NE Buffer 2 (NEB) using a thermocycler (Long Gene, A200, China). The PCR products were digested with T7EN1 for 30 min at 37°C and then separated on a 1.5% agarose gel. The PCR products with mutations detected by the T7EN1 cleavage assay were then sub-cloned into a PMD-18T vector (Takara, Japan).

Lentivirus generation and cell infection
The resulting shuttle vectors with or without sgW(7+8) were mixed with a packaging mix (pMD2.G and PsPAX2). Then, the mixed vectors were added to the opti-MEM (Invitrogen, Carlsbad, USA) culture medium and transfected into the 293T packaging cell line. Recombinant lentivirus expressing both GFP and sgRNAs of Wisp2 were harvested and filtered through a 0.45μm filter (Millipore, USA) after 48 hours or 72 hours. For the infection, the cells were transduced with lentiviruses in 5% FBS growth media supplemented with polybrene (8μg/ml). The cells were incubated overnight with the lentivirus and cultured in fresh 5% FBS growth media for an additional 3 days. Subsequently, the cells were cultured in 10% FBS growth media. Finally, we detected the cutting efficiency as described above.

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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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