pSPCas9(BB)-2A-GFP plasmids

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

Clonal Cell Lines Carrying Deletions in Candidate Genes:
Clonal knockout NIH/3T3 lines carrying deletions in candidate genes were generated using a dual sgRNA strategy. Briefly, two sgRNAs targeting candidate genes with an interval spanning anywhere from 50-600 bases (see Table S3) were designed using the CRISPR Design Tool (http://crispr.mit.edu) and cloned into pSpCas9(BB)-2A-GFP (PX458; Addgene #48138; (Ran et al., 2013)) and pSpCas9(BB)-2A-mCherry, the latter generated by replacing the GFP cassette in PX458 with mCherry. Five days after co-transfection into NIH/3T3 cells using X-tremeGENE 9 (Roche), GFP and mCherry double positive single cells were sorted into a 96-well plate using FACSAria II. Clonal lines were screened by PCR to detect excision of the genomic DNA between the two sgRNA cut sites (gels showing successful editing of candidate genes are shown in Table S3). When possible, knockout clones were further confirmed by Western Blotting using commercially available antibodies (Rab34, Pdcl, and Mgrn1). Clonal knockout mESCs were generated using the same two sgRNAs for each gene used in NIH/3T3 cells (see Table S3). For mESCs, sgRNAs were cloned into plasmid PX459 (Addgene #48139; (Ran et al., 2013)). Plasmids were electroporated into mESCs using the Lonza nucleofection system (Nucleofector 2b Device using the program A-023 and Lonza Cell Nucleofector Kit #VAPH-1001). mESCs were cultured under feeder free conditions in 2i media (Dulbecco’s Modified Eagle’s Medium F12 (Gibco) and Neurobasal Medium (Gibco) (1:1 ratio) supplemented with N-2 Supplement (Gibco), B-27 Supplement (Gibco), 1% penicillin/streptomycin (Gemini Bio-Products), 2mM L-glutamine (Gemini Bio-Products), 40 μg/ml Bovine Serum Albumin (Sigma), 55 μM 2-mercaptoethanol (Gibco), 5 μM CHIR99021 (Axon), 1 μM PD 98059 (Axon), and 1000 U/ml ESGRO LIF (Millipore)). 24 h post nucleofection, cells were selected with 1.5 μg/ml puromycin for 48 h. One week later, individual mESC colonies were manually picked, expanded, and screened by PCR to detect deletion of the genomic DNA segment between the two guides (see Table S3).

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Manufacturer protocol

Download the product protocol from Addgene for pSPCas9(BB)-2A-GFP plasmids below.

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