自韩春雨在NBT上发表NgAgo基因编辑技术以来，事情几经翻转，几乎一直是科研圈的关注热点。韩春雨本人也遭受巨大质疑，眼看就要朝着中国版小保方晴子发展，这不，我们亲爱的韩博士终于向Addgene提供了更加详细的操作protocol。在这篇protocol中韩博士对诸多小细节进行了特别提示。不过助手君还注意到本篇protocol中多处提到pH=8.0，之前便有网友向助手君表示，纯化NgAgo的菌就是一种噬碱菌，所以NgAgo极有可能得在碱性环境下才能发挥最好活性，这篇protocol似乎正印证了这点。所以已经投入了巨大经费的不妨先按照此篇protocol操作看看，也许真的可以呢？我们静待各位佳音！

1、细胞培养
293T细胞使用含10%FBS（Gibco）和双抗（penicillin/streptomycin ）的高糖 DMEM（Gibco）于5% CO2 的37°C培养箱中培养。当细胞密度达到约60%时进行转染。细胞转染前需要将培养基换成含2% FBS（热失活）的新鲜培养基。293T细胞贴壁不牢，换液需要小心轻柔，不要将细胞吹起。

2、转染
2.1 使用Promega试剂盒抽提NLS-NgAgo表达质粒，并将终浓度稀释到100ng/μl（用水稀释，水用NaOH将pH调至8.0）。
2.2 5’ 磷酸化的向导ssDNA用水（pH=8.0）溶解至终浓度100ng/μl。以24孔板一孔为例：将200-250ng NLS-NgAgo表达质粒和100-300ng向导DNA稀释于50μl Opti-MEM (Gibco)中；并将1.25μl Lipofectamine® 2000稀释于50μl Opti-MEM中，将两种混合液孵育5min。
2.3 将上述两种预混液混在一起并轻轻吹匀，室温孵育20min。之后将其加入细胞培养板中。

第2点备注
*由于NgAgo遵循“one-guidefaithful”的原则，也就是说，向导只有在NgAgo表达的时候才会被装载，所以有时候多次转染gDNA可以提高NgAgo装载gDNA的效率（例如：根据不同细胞内NgAgo的不同表达节点，在初次转染之后可以选择在8、12或者24h时进行二次转染）。
*在下面的步骤中，转染后48-60h之间收细胞，此时细胞密度在90%比较理想。细胞过密会影响基因组编辑效率。比如，HDR就只能发生在S期和G2期之间。

3、 基因组DNA编辑
3.1 细胞转染后48-60h，胰酶消化收集细胞。将四孔细胞收集到一个1.5 ml EP管中。
3.2 对于基因组DNA的抽提，需要在每管中加入500 μl细胞裂解缓冲液(50 mM Tris，100 mM EDTA，0.5% SDS，pH 8)和10 μl proteinase K(10 mg/ml)，轻轻充分混匀。将其置于55℃孵育2h。
3.3 每管加入200μl Tris-Phenol（Tris饱和酚）and 200 ul trichloromethane（氯仿），轻轻充分混匀。孵育5 min后，12,000 rpm离心15min以分离水相和酚相。
3.4 将水相小心收集如一个新的EP管中。
3.5 重复步骤3-3和3-4一次并收集水相。
3.6 加500μl氯仿到收集水相的EP管中，轻轻充分混匀，静置5min，之后12,000rpm离心15min，将水相与酚相分离。小心将水相转移至一个新的干净EP管中。
3.7 重复步骤3-6。
3.8 向收集管中加入900 μl的乙醇，并在-20℃孵育30 min。
3.9 12,000 rpm离心10 min，用500 μl乙醇洗涤DNA沉淀三次。
3.10 晾干DNA沉淀，之后加入50 μl 0.5 x TE，并将基因组DNA稀释至100ng/μl以便后期使用。

注意事项：
1. NgAgo/gDNA系统对胞内细菌（包括支原体）污染十分敏感，而这些污染往往广泛存在并且无法用肉眼观察到（助手君按：所以细胞要做支原体检测，这个污染之事韩春雨在贴吧等回应中反复强调过，不过支原体污染确实在国内广泛存在）。所以实验之前必须确保细胞无污染。
2. 因为Agos需要Mg2+，所以在消化和铺细胞时不要用含EDTA的试剂（注：很多Trypsin中含有EDTA）。可以选择补充Mg2+至终浓度5 mM（根据实验细胞类型进行适当调整）。
3. 由于P3000会干扰ssDNA的转染，所以不要使用Lipo 3000（大写的汗(⊙﹏⊙)b）。Lipo2000是一个很好的选择。诸如电转之类的其他转染方法有待测试。
4. 建议用T4 PNK(Biolab)对 ssDNA 进行5’ 磷酸化。

英文原文：
1. Cell culture
293T cells are maintained in high-glucose DMEM (Gibco) supplemented with 10% FBS(Gibco) and penicillin/streptomycin at 37°C with 5% CO2 incubation. When cells reach their ≈60%confluence, perform transfection. Before transfection, cells are changedto medium containing 2% FBS（heat inactivated）. Since 293T cells are not firmly attached, be gentle and do not disturb cells when changing medium.

2. Transfection
2-1 NLS-NgAgo expressing plasmid is extracted with Wizard® Plus SV Minipreps DNA Purification System (Promega), and is adjusted to 100 ng/μl in water (pH 8.0, alkalization by NaOH).
2-2 5’ phosphorylated ssDNA guides are dissolved to 100 ng/μl in water (PH 8.0) For each well of a 24-well plate, 200-250 ng NLS-NgAgo expression plasmidand 100-300 ng guides are diluted in 50 μl Opti-MEM (Gibco); 1.25 μl Lipofectamine®2000 is diluted in 50 μl Opti-MEM. Incubate the DNA mix and lipofectamine mix for 5 min.
2-3 Combine the DNA mix and lipofectamine mix with gentle pipetting and incubatefor 20 min. The DNA/lipo mixture is then added into each well of cells.

*SinceNgAgo follows “one-guide faithful” rule, i.e. guides can only be loaded when NgAgo protein is in the process of expression, to improve the efficiency ofgDNA loading to NgAgo, sometimes multiple transfection of gDNA helps (e.g. depending on the different kinetics of NgAgo expression in different cells, a second transfection of gDNA can be performed 8, 12 or 24 hours after the primary transfection)
*As stated below, cells will be harvested 48-60 hours after transfection. 90% confluence of the cells on harvesting is ideal. Cell overplating significantly weakens the efficacy of genome editing. Taking HDR as anexample, it occurs only during S and G2 phases.

Note:
1. NgAgo/gDNA system is extremely sensitive to contamination of intracellular bacteria(including mycoplasma) which are widespread and leave no visible signs of presence. Carefully excluding the presence of intracellular bacteria before performing experiments.
2. Because Agos need magnesium, avoid EDTA when detaching and seeding cells into the plates for transfection. Alternatively, supplementing Mg2+ to 5 mM (may need optimization to your cell type).
3. Avoidusing Lipofectamine® 3000 since the supplement P3000 interferes with ssDNA transfection. Lipofectamine® 2000 is a good choice. Other transfection methods such as electroporation are yet to be tested.
4. Ideally, 5' phosporylation of ssDNA guide by using T4 PNK (Biolab):
T4 PNK 2μl
T4 ligase buffer (containing ATP): 12 μl
1OD ssDNA (about 33 μg) in H2O: 100 μl
(alternatively)additional ATP (25 mM) 2 μl
Add H2O to a final volume of 120μl
Incubate at 37℃ overnight
After 5' phosphorylation, the resultant ssDNA needs no purification, dilute by water(PH8) to 300 μl with a final concentration of 10 nM or 100 ng/μl)

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