pSpCas9(BB)-2A-Puro (PX459) V2.0

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

CRISPR/cas9-mediated gene knockout in 3T3-F442A cells
Guide RNA sequences targeting SWELL1 gene were designed using web based CRISPR design tool (http://crispr.mit.edu/) (Supplementary Table 4) and were cloned in a Cas9 expressing bicistronic vector (pSpCas9(BB)-2A-Puro) as described59. 3T3-F442A cells were either nucleofected with single (399 for KO1) or double (295/399 for KO2) guide vectors using Amaxa® Cell Line Nucleofector® Kit V per manufacturer’s instructions. Puromycin selection medium (1 μg/ml) was added 48 hours after nucleofection and maintained for 5 days. The cell pool was expanded and clonally diluted. Genomic modifications of individual clones were confirmed by Sanger sequencing for KO1, whereas a PCR screen was used to confirm the deletion of the gene fragment between the two guide pairs for KO2. Control 3T3-F442A adipocytes were either WT 3T3-F442A adipocytes or 3T3-F442A clonal lines isolated from the cas9/gRNA transfections that did not undergo CRISPR/cas9-mediated gene editing.

Generation of WT/GRB2KD and SWELL1 KO/GRB2 KD 3T3-F442A adipocytes
Lenti GRB2 and LUC (luciferase) shRNAs, packaging, and envelope plasmids are kind gifts from Dr. Jon C. Houtman at The University of Iowa. The LUC shRNA was used as a control. Viral production and transduction were performed as described in Bilal et. al60. In brief, lentiviral vector plasmid, packaging, and envelope plasmids were transfected into 293T cells. Virus containing supernatant was harvested and used to transduce WT and SWELL1 KO 3T3-F442A cells. Cells were incubated with virus in the presence of 8 μg/ml Hexadimethrine bromide (Sigma) for 72 hours. Post transduction, the cells were selected with 0.5 μg/ml puromycin. Puromycin was then gradually increased to 2 μg/ml (0.5 μg/ml increments). Non-transduced cells were used as selection control. Selection was maintained for an additional week after the selection control cells were 100% sloughed off. The transduced cells were then cultured and differentiated as described in cell culture.

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Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-Puro (PX459) V2.0 below.

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