dCas9-SAM技術應用詳解以及慢病毒包裝詳細操作手冊

在之前的文章《一文教會你利用CRISPR-dCas9技術調控內源基因的轉錄激活與抑制》我們為大家講解了利用CRISPR-dCas9技術調控內源基因激活的技術，今天我們再次邀請到領域內的牛人為我們講解dCas9-SAM相關技術並為大家呈上慢病毒包裝的詳細操作手冊。如需獲取本文相關質粒請按文末要求添加小編微信，還可以加入CRISPR-Cas9技術專題群討論交流學習。

dCas9-SAM: an engine for activation the endogenous gene expression

Synergistic activation mediator (SAM)is developed at the basis of CRISPR/Cas9 technique. Wildtype cas9 has been mutated at amino acid positionsD10AandH840Awhich completely inactivate both the nuclease and nickase activities. The resulted double mutated cas9 is called asdcas9. With the help of a target-specific guide RNA (sgRNA), it can bind to a specific region of DNA without cutting it. In this context, dCas9 carries a C-terminal tripartite synergistic activation mediator (SAM). By targeting dCas9-SAM to a promoter region, we can specifically induce expression of virtually any gene of interest.

Plasmids for this protocol

The SAM system involves three lentivirus plamids.Lenti sgRNA(MS2) purohave BsmBI restriction enzyme site, which is for insertion of the oligo sgRNA under the hU6 promoter. The result sgRNA(MS2) lentivirus express the fused MS2 and specific sgRNA while gives the cells the puromycin resistance.Lenti dCas9-Vp64has fused dcas9 and vp64 cassette drived by EF1α promoter and has the Blastcidin resistance. The third plamids for SAM system islenti MS2-p65-HSF1, while it has a distinct hygromycin resistance genes. To sum up, SAM system is widely used for the activation of interest gene by targeting the activation regulatory factors to the promoter the region. It needs to packet three lentivirus harboring the all components of the system with distinct resistance genes.

Procedure:

1. Design the sgRNA_SAM at the follwing website

Optimized sgRNAs for any coding gene can be found using SAM Cas9 activator design tool designed by ZhangLab, MIT at the following website:

http://sam.genome-engineering.org/database/

Synthesize two oligos of the following form.

Example for sgRNA oligo design: Note that the NGG PAM is not included in the designed oligos.

Tips: Standard de-salted oligos (usually the most inexpensive synthesis) are sufficient for cloning. If not already resuspended, dilute each oligo to 100 μM in sterile water or TE.

2. Insert the SgRNA oligo into lenti sgRNA(MS2)_puro plasmid, the map of lenti sgRNA(MS2)_puro is as follows:

(1). Oligo annealing

Mix the components above and anneal in a thermal cycler with the following conditions:

On thermal cycler with heat lid.

37 ℃, 30 min

95 ℃, 5 min

Ramp down to 25℃ at 0.1℃/sec. (when step 25℃ is high lighted, click option, set Ramp as 0.1℃/sec)

25 ℃, 5 min

4 ℃, 5 min

Put on ice, or keep in -20℃ freezer for storage.

(2). Digest lenti sgRNA(MS2)_puro plasmid with BsmBI

Tips: Purify the enzyme digested product with PCR product purification kit, and make sure that the concentration is more than 50 ng/μl.

3. Ligation

Dilute the annealed target oligos 200 times for further ligation

Ligate for more than 2 hours at 22℃. (Overnight is better.)

4. Transformation and identification of positive clones

Transform the ligation product into the stabl3 competence cells and pick single clones and culture for several hours in 37℃ incubator, and perform PCR to identify the positive clones.

sgRNA(MS2)-F: GAG GGC CTA TTT CCC ATG ATT CCT TCA TAT

Use Oligo-reverse and sgRNA(MS2)-F as primers, and the PCR product is about 350 bps.

Note: sgRNA(MS2)-F is the sequencing primer for oligo sgRNA

5. Plasmid extraction and sequencing validation

Culture the positive clone in 10 ml Amp+ LB medium, and extract plasmid. Use sgRNA(MS2)-F as sequencing primer.

6. Lentivirus production

Lentiviral Production Protocol

The day before transfection:

The day before transfection, seed around 5×106 HEK293FT cells per 100mm petri dishes.

The day of transfection:

1. Check to make sure cells are at least 70% confluent before transfection.

2. Combine the appropriate amount of plasmid DNAs in a 1.5 ml conical tube:

For per100mm petri dish:

10 μg Lentivirus backbone plasmid harboring inteterest gene

4 μg pdR8.2. (structural vector)

2 μg pMDG2 (envelope vector)

16 μg in total

3. Add filtered water to a final volume of 878 μl.

4. Add 122 μl 2M CaCl2and mix.

5. Drop 1000μlof 2×HBS to the abvove mixture slowly. Note: Shaking the tube continuously to avoid generating large particles

6. Sit mixture at room temperature for 2 min.

7. Remove old media and replace with 9 ml new complete culture media.

8. Add all of mixture to the cells gently and evenly. Swirl the petri dish to distribute evenly and put it back to incubator.

9. 8 hours later, Change media to remove the transfection reagent and replace with 12 ml new complete culture media.

10. Generally, 72 hours after transfection , harvest cell culture supernatant containing lentivirus and filter through a 0.45μm filter.

11. Concentrate the lentivirus with PEG8000 or with ultra-speed centrifugation.

Transfection reagent stock preparation:

2 MCaCl2

2×HBS:

50mM HEPES

10mM KCl

12mM Dextrose

280mM NaCl

1.5mM Na2HPO4

Resuspend in 900 mlH2O, adjust pH to 7.04 (with NaOH if using HEPES free acid, with HCl if using HEPES sodium salt). Raise volume to 1000 ml and carefully re-adjust final pH to 7.05 exactly (Note: pH needs to be very precise at pH 7.04 – 7.05). Filter through 0.22μM and store in aliquots at -20°C (good for up to 6 months).

Suggestions and Tips: You can also choose other transfection reagents including lipofectamine2000, PEI and Turbofect (Thermo/Fermentas). Remember that the transfection efficiency should be over 90 percent.

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