Supplementary MaterialsSupplementary document 1: PCR primers for target loci amplification and indexing for deep sequencing of on-target and off-target sites. occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells. DOI: http://dx.doi.org/10.7554/eLife.04766.001 Cas9 used in this study carries at C-terminus an HA tag and two nuclear localization signal peptides which facilitates transport across nuclear membrane. The protein was expressed with a N-terminal hexahistidine tag and maltose binding protein in Rosetta 2 cells (EMD Millipore, Billerica, MA) from plasmid pMJ915. The His tag and Leupeptin hemisulfate maltose binding protein were cleaved by TEV protease, and Cas9 was purified by the protocols explained in Jinek et al., 2012. Cas9 was stored in 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) at pH 7.5, 150 mM KCl, 10% glycerol, 1 mM tris(2-chloroethyl) phosphate (TCEP) at ?80C. In vitro T7 transcription of sgRNA The DNA template encoding for any T7 promoter, a 20 nt target sequence and an optimized sgRNA scaffold (Chen et al., 2013) was put Leupeptin hemisulfate together from synthetic oligonucleotides (Integrated DNA technologies, San Diego, CA) by overlapping PCR. Briefly, for the EMX1 sgRNA template, the PCR reaction contains 20 nM premix of BS16 (5- TAA TAC GAC TCA CTA TAG GTC ACC TCC AAT GAC TAG GGG TTT AAG AGC TAT GCT GGA AAC AGC ATA GCA AGT TTA AAT AAG G -3) and BS6 (5- AAA AAA AGC ACC GAC TCG GTG CCA CTT TTT CAA GTT GAT AAC GGA CTA GCC TTA TTT AAA CTT GCT ATG CTG TTT CCA GC -3), 1 M premix of T25 (5- TAA TAC GAC TCA CTA TAG -3) and BS7 (5- AAA AAA AGC ACC GAC TCG GTG C -3), 200 M dNTP and Phusion Polymerase (NEB, Ipswich, MA) according to manufacturer’s protocol. The thermocycler setting consisted of 30 cycles of 95C for 10 s, 57C for 10 s and 72C for 10 s. The PCR product was extracted once with phenol:chloroform:isoamylalcohol and then once with chloroform, before isopropanol precipitation overnight at ?20C. The DNA pellet was washed three times with 70% ethanol, dried by vacuum and dissolved in DEPC-treated water. The DYRK1 sgRNA template was put together from T25, BS6, BS7 and BS14 (5- TAA TAC GAC TCA CTA TAG GTT CCT TAA ATA AGA Take action TTG TTT AAG AGC TAT GCT GGA AAC AGC ATA GCA AGT TTA AAT AAG G -3). The CXCR4 sgRNA template was put together from T25, SLKS3 (5- TAA TAC GAC TCA CTA TAG GAA GCG TGA TGA CAA AGA GGG TTT TAG AGC TAT GCT GGA AAC AGC ATA GCA AGT TAA AAT AAG G -3), SLKS1 (5- GCA CCG Take action CGG TGC CAC TTT TTC AAG TTG ATA ACG GAC TAG CCT TAT TTT AAC TTG CTA TGC TGT TTC CAG C -3) and SLKS2 (5- GCA CCG Take action CGG TGC CAC TTT TTC AAG -3). An 100-l T7 in vitro transcription reaction consisted of 30 mM TrisCHCl (pH 8), 20 mM MgCl2, 0.01% Triton X-100, 2 mM spermidine, 10 mM fresh dithiothreitol, 5 mM of each ribonucleotide triphosphate, 100 g/ml T7 Pol and 1 M DNA template. The reaction was incubated at 37C for 4 hr, and 5 models of RNase-free DNaseI (Promega, Madison, WI) was added to digest the DNA template 37C for 1 hr. The reaction was quenched with 2xSTOP answer (95% deionized formamide, 0.05% bromophenol blue and 20 mM EDTA) at 60C for 5 min. The RNA was purified by electrophoresis in 10% polyacrylamide gel made up of 6 M urea. The RNA band was excised from your gel, grinded up in a 15-ml tube, and eluted with 5 vol of 300 mM sodium acetate (pH 5) overnight at 4C. One equivalent of isopropanol was added to precipitate the RNA at ?20C. The RNA pellet was collected by centrifugation, washed three times with 70% ethanol, and dried by vacuum. To refold the sgRNA, the RNA pellet was first dissolved in 20 mM HEPES (pH 7.5), 150 mM KCl, 10% glycerol and 1 mM TCEP. The sgRNA was heated to 70C for 5 Leupeptin hemisulfate min and cooled to room heat. MgCl2 was added to a final concentration of 1 1 mM. The sgRNA was again heated to 50C for 5 min, cooled to room temperature and kept on ice. The sgRNA concentration was determined by OD260nm Rabbit polyclonal to BMP2 using Nanodrop and adjusted to 100 M using 20 mM HEPES (pH 7.5), 150.