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

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Protocol tips
There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

Cloning
sgRNAs targeting , , and were designed using the online CRISPR design tool at . The selected sgRNA sequences are listed in . The sgRNA and SpCas9 expression cassette from PX458 (pSpCas9(BB)-2A-GFP) or PX459 (pSpCas9(BB)-2A-Puro) (both plasmids were a gift from Dr. F. Zhang; Addgene, plasmids: # 48138 and # 62988 []) were cloned into the baculovirus donor plasmid pAceBac1 (a kind gift from Dr. Imre Berger, EMBL, Grenoble, France [, ]) from which the promoter was removed. Where applicable, the HDR template was introduced into a unique NotI site of the same donor vector. The HDR template for creating Aurora B H250Y consists of a 312 bp sequence ranging from chr17:8205171–8205482 (GRCh38) containing a mutation encoding H250Y and two additional silent mutations to prevent recognition by the sgRNA.

Baculovirus production
Baculovirus was produced by transfection of bacmids into Sf9 cells. Cells were seeded in 6 well plates (0.5*10^6 cells/well), followed by transfection using FuGENE HD (Promega, Madison, WI, cat. no. E2311). P1 virus was harvested after 3–4 days and subsequently used to transduce 20 ml suspension cultures of early log phase Sf9 cells (1–2*10^6 cells/ml). P2 virus was harvested 3–4 days after transduction. Viral titers were determined using an end point dilution assay.

Viral transduction
For baculoviral transduction, cells were trypsinized and taken up in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, cat. no. R0883) supplemented with 10% heat inactivated FBS, 2 mM UltraGlutamine 100 units/ml penicillin and 100 μg/ml streptomycin, hereafter called RPMI-1640. The cells were then pelleted (5 min. at 483 x g) and re-suspended to a concentration of 75.000–200.000 cells/ml in RPMI-1640. Virus was added to the cell suspension at an MOI of 25–75 and cells were subsequently plated. For RPE-1 and MCF10A cells, the medium was changed back to their normal culture medium after 16 hours.

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Papers

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Manufacturer protocol

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

Download manufacturer protocol

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