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

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Publication protocol

knockout using the CRISPR-Cas9 system
Three single guide RNA sequences (plasmids UK1448, UK1449 and UK1450; ) for targeting the third exon of (Chinese hamster) were selected from the CRISPy database [URL: , ()] and duplex DNA oligonucleotides of the sequences were inserted into the pSpCas9(BB)-2A-GFP plasmid (plasmid UK1359; ) following published procedures (). 2 x 10 CHO-K1 or CHO-K1 reporter cells () were plated in 6-well plates. Twenty-four hr later the cells were transfected with a combination of guide RNA/Cas9 plasmids UK1448 and UK1449 or UK1448 and UK1450 (2 µg total plasmid DNA per transfection) using Lipofectamine LTX (Invitrogen). Thirty-six hr after transfection the cells were washed with PBS, resuspended in PBS containing 4 mM EDTA and 0.5% (w/v) BSA, and GFP-positive cells were individually sorted by fluorescence-activated cell sorting (FACS) into 96-well plates using a MoFlo Cell Sorter (Beckman Coulter). Clones were then analyzed by a PCR-based assay to detect mutations as described (). Briefly, primers were designed for the region encompassing the RNA guide target sites and the reverse primer was labeled with 6-carboxyfluorescein (6-FAM) on the 5’ end. A PCR reaction was set up using 5 µl of AmpliTaq Gold 360 Master Mix (Applied Biosystems, UK), 0.6 µl of a mix of 10 µM forward and labeled reverse primers, 3.4 µl HO and 1 µl genomic DNA (approximately 10 ng/µl). PCR was performed as follows: 95°C for 10 min, 10 x (94°C for 15 s, 59°C for 15 s, 72°C for 30 s), 20 x (89°C for 15 s, 59°C for 15 s, 72°C for 30 s), 72°C for 20 min. PCR products were diluted 1:100 in water and fragment length was determined on a 3130xl Genetic Analyzer (Applied Biosystems) and the data were analyzed using the Gene Mapper software (Applied Biosystems). Clones for which frameshift-causing insertions or deletions were detected for both alleles were sequenced to confirm the mutations.

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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