Rapid Assembly and Cloning of Zinc Finger Proteins with Multiple Finger Modules

Experimental

PCR Amplification. For convenience and efficiency of the vector PCR, the vector template DNA was constructed into pJHU-1 vector (a gift from professor Srinivasan Chandrasegaran at department of Environmental Health Sciences of the Johns Hopkins University), a 2-kb minimal size cloning vector. Two ZF template oligonucleotides and all PCR primers were purchased from Bioneer Cooperation. For a typical PCR reaction, the reaction volume was 50 μL containing 5 μL of the 10X Pfu buffer, 4 μL of dNTP (2.5 mM each), 1 μL of 10 μM primer mix, 1 μL of 50 ng/μL template DNA and 1 μL of Pfu polymerase (5 U/μL) (Labo Pass). For vector PCR, the reaction mixture was incubated at 94 ℃ 5 min, and 30 cycles of denaturation (94 ℃ for 1 min), annealing (57 ℃ for 1 min) and extension (72 ℃ for 4 min) were performed. And, the reaction mixture was kept at 72 ℃ for 10 min. For the ZF PCR, the reaction condition was same except the extension time, which was 1 min. After PCR, reactions were subjected to DpnI digestion at 37 ℃ for 4 h to remove template plasmid DNAs, if necessary. Each PCR product was subjected to electrophoresis and size-checked in agarose gel.

Gibson Assembly and Cloning. Vector DNA fragments were gel-isolated by MEGA quick-spinTM total kit (iNtRON). The gel-isolated vector fragments and two (or three) PCR products of ZF modules for 3 (or 4)-finger ZFP construction. For the Gibson assembly, Gibson AssemblyTM master mix was used (New England Biolab). In this assay, the reaction volume was 20 μL containing 10 μL of the 2X Gibson Assembly Master Mix and PCR-amplified DNA fragments, 0.2 pmole of vector fragment and 0.6 pmole of each ZF module fragment. The reaction was incubated for 1 h at 50 °C. After incubation, the DNA was used for transformation by electroporation. For expression of CCR5-L and -R ZFN, the constructed ZFP genes was transferred into pET28a vector (Novagen) using NcoI and HindIII sites.

DNA Cleavage Activity Assay CCR5-L-ZFN and CCR5- R-ZFN cloning vectors were used for in-vitro translation reactions by TNT® T7 Quick-Coupled Transcription/Translation System (Promega). In this assay, reaction volumes were 20 μL containing 2 μL of the 10X reaction buffer (50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT), 500 ng of the ScaI-linearized target DNA, and 2 μL of each expressed either CCR5-L or -L- ZFN. The reaction was incubated for 12 h at 22 ℃. After the DNA cleavage, 10 μg of RNase A (GENE ALL) was added into the reactions and incubated for 30 min at 22 ℃ to remove RNAs. For removal of proteins in the reaction, 10 μg of Protease K (New England Biolab) was added to the samples and incubated for 2 h at 55 ℃. The reaction was subjected to agarose gel electrophoresis and stained with ethidium bromide. And DNAs were visualized under UV.

Supporting Information. Supplementary data associated with this article can be found in the online version.