Creation of TALE nucleases
Construction of AAVS1-L, AAVS1-R, MSTN-L, and MSTN-R for co-transfection was previously described . AAVS1-L/AAVS1-R and MSTN-L/MSTN-R for creation of pTAL10-AAVS1 and pTAL10-MSTN, respectively, was constructed using Golden Gate TALEN Kit 2.0 (Kit #1000000024) from Addgene. These sequences were then added to pTAL10 (Figure 2) by using BsmBI and BsmBI/BsaI digestion respectively. Further details on creation of TALE nucleases using pTAL10 scaffold is discussed below and in the supplemental protocol.
Cell culture and transfection
HEK293 cells were cultured in DMEM supplemented with 10% FBS, 1% L-glutamine (Thermo Scientific Hyclone, Waltham, MA), and 1% Penicillin-Streptomycin (Thermo Scientific Hyclone, Waltham, MA). Transient transfection by Xfect transfection reagent (Clontech, Mountain View, CA) was done according to manufacturer’s recommendation. In short, HEK293 cells for each downstream application (genomic DNA or protein extraction) were seeded in a 6-well plate until 60-70% confluency. Thereafter, 5 μg of total DNA (pTAL10-TALEN single plasmid transfection, or TALEN-L/TALEN-R co-transfection) in nanoparticle complex solution was added to the culture medium dropwise, followed by replacement of medium 16–20 hours post transfection. After 72 hours, either genomic DNA or protein was extracted from these cell lines. Transfection efficiency was determined by fluorescence microscopy analysis: 1) the average percentage of mCherry- or GFP-positive cells for co-transfection or 2) the percentage of GFP-positive cells for pTAL10-TALENs.
HEK293 transfected with single pTAL10-MSTN or co-transfection with MSTN-L and MSTN-R were lysed 48–72 hours post transfection with cold RIPA buffer supplemented with protease inhibitors (1 mM PMSF and 1 mM Benzamidine). Protein concentration was determined by using Bradford Protein Assay Kit (IBI Scientific, Peosta, IA). 20 μg of protein were resolved on a 4-20% Mini-PROTEAN TGX Precast Gel (Bio-Rad Laboratories, Hercules, CA) and transferred onto a PVDF membrane (Millipore, Billerica, MA). Immunoblotting was done with the following antibodies: mouse monoclonal anti-FLAG (F3165; Sigma-Aldrich, Saint Louis, MO), polyclonal anti-HA-Tag antibody (Clontech, Mountain View, CA), mouse monoclonal anti-GAPDH (Millipore, Billerica, MA), and HRP conjugated goat anti-mouse IgG (Millipore, Billerica, MA). Membranes were developed using ECL 2 Western Blotting Substrate (Pierce Biotechnology, Rockford, IL) and images were captured using ChemiDoc XRS + system (Bio-Rad Laboratories, Hercules, CA).
T7E1 mismatch-detection assay
Cleavage activity of AAVS1 and MSTN TALENs, as measured by small deletion/insertion mutations (indels), were quantified by mismatch-recognizing T7E1 as described previously . Briefly, cells transfected with respective TALENs had genomic DNA extracted 48–72 hours post-transfection. Amplification was achieved by using AccuPrime Pfx (Invitrogen, Carlsbad, CA). PCR primers were designed to amplify site surrounding human AAVS1 or human MSTN locus. Specifically, the primer pair for human MSTN binds to Intron 1–2 (hGDF8-F1, 5′-TGGAGGGGTTTTGTTAATGG-3′) and Intron 2–3 (hGDF8-F2, 5′-TATTGGGTACAGGGCTACCG-3′). The primer pair used for AAVS1 targets the following sites: AS1L-833 F (5′- TTCAGGTTCCGTCTTCCTCCACTC – 3′) and AS1l-1998R (5′- AACTGACGCACGGAGGAACAAT-3′). The DNA fragment was purified by using Wizard SV Gel and PCR Clean-Up (Promega, Madison, WI), followed by measurement of DNA concentration by using NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmingdon, DE). 600 ng of DNA for MSTN targeting TALEN or 200 ng of DNA for AAVS1 in NEBuffer 2 (New England BioLabs, Ipswich, MA) was then denatured at 95°C for 10 minutes, and reannealed slowly using the following temperature program to form DNA heteroduplex if NHEJ occurred: 90 cycles of 95-59°C with a 0.4°C decrease per cycle for 20 seconds, 90 cycles of 59 to 32°C with a 0.3°C decrease per cycle for 20 seconds, and 20 cycles of 32 to 26°C with a 0.3°C decrease per cycle for 20 seconds. 0.5 uL T7 Endonuclease I (T7E1; New England BioLabs, Ipswich, MA) was added to the reannealed DNA samples, followed by incubation for 30 minutes. DNA samples were then subjected to electrophoresis on a 2% TAE agarose gel containing Gel-Red (Biotium, Mayward, CA). The gels were imaged using ChemiDoc XRS + system (Bio-Rad Laboratories, Hercules, CA), and densiometry of DNA bands were quantified using ImageJ software (NIH, Frederick, MD). Mutation frequencies were calculated using the formula: fractional modification = 1-(1-(total densiometry of fraction cleaved/total densiometry))0.5 as described .
Fluorescence and bright-field images were taken as previously mentioned . In short, images were taken using an inverted Nikon Ti-E microscope equipped with a Xenon lamp (Hamamatsu Photonics Systems, Bridgewater, NJ), a 40 × 1.30 NA objective (Nikon, Tokyo, Japan), and an Evolve 512 EMCCD camera (Photometrics, Pleasanton, CA). The EMCCD camera was cooled to -80°C during imaging. Images were documented and analyzed using NIS-Elements Advanced Research software package (Nikon, Tokyo, Japan).
All data were expressed as mean ± SEM. Statistical differences were determined by unpaired Student’s t-test for two groups using GraphPad Prism 5.0. Statistical significance was defined to be p < 0.05.