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

CRISPR Human - Deletion AURKB

Experiment
CRISPR Human - Deletion AURKB
Product
pSpCas9(BB)-2A-GFP (PX458) from Addgene
Manufacturer
Addgene

Protocol tips

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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Discussion

Discussion

1 year ago

Author: Milena Alexeyeva Russian Federation

DNA insert using CRISPR

I would like to excise a large strand of DNA and insert a new one using CRISPR. My problem is that my strand will be a little over 1kb and I am not sure if this is going to be a limiting factor. Also, how long should the homology arms be for a region of this size?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Deletion AURKB using pSpCas9(BB)-2A-GFP (PX458) from Addgene.

Paper title
Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells
Full paper
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Manufacturer protocol

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

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Videos

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