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

Protocol tips

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	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

Generation of ZPR9 (T252A) knockin (KI) and knockout (KO) cell lines
Genomic mutations in 3T3-L1 or HEK293 cells were generated using the CRISPR/Cas9 system as described previously30,31. In brief, single-guide (sg) RNAs were designed to target the genomic areas adjacent to the ZPR9 mutation site (target sequence, 5′-GCTGAGGAAGGCCCACCCCT-3′). Two complementary oligos (5′-CACCGGCTGAGGAAGGCCCACCCCT-3′ and 5′-AAACAGGGGTGGGCCTTCCTCAGCC-3′) containing the ZPR9 guide sequence and Bbs1 ligation adapters were synthesized by Bioneer Ltd. (Cheongwon, Korea). The annealed oligos were ligated into the Bbs1-digested pX458 vector (Addgene plasmid no. 48138) using the Quick-Ligation system (New England BioLabs, Ipswich, MA, USA). To generate the ZPR9 (T252A) KI cell line, 3T3-L1 cells were cultured in a 24-well plate at ~60% confluence and cotransfected with 1 μg of ZPR9 sg RNA plasmid and pUC19 ZPR9(T252A) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) or WelFect-Ex™ Plus (WelGENE, Daegu, Korea). To generate the ZPR9 KO cell line, HEK293 cells were transfected with 1 μg of ZPR9 sg RNA plasmid without pUC19. Forty-eight hours after transfection, the cells were trypsinized and diluted in medium for single-cell seeding in a 96-well plate, and GFP-positive cells were screened, followed by genomic DNA extraction. Exon 2 of ZPR9 was amplified by PCR using a ZPR9-specific PCR primer pair as follows: 5′-ATTGGGAAGATATTGATTCTGATGAAGA-3′ (forward) and 5′-CCAAGTATTTAATCAGT CCCTTAATATCTG-3′ (reverse). The PCR products were A-tailed and cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). The individual ZPR9 (T252A) KI clones were then confirmed by DNA sequencing (Bioneer Ltd., Cheongwon, Korea). On the other hand, the ZPR9 KO clones were validated by immunoblotting with an anti-ZPR9 antibody.

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Manufacturer protocol

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

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