Paired Guide RNAs/nickase All-in-one system in lentivirus backbone to minimize the off-target effect
CRISPR/ Cas9 technique, as the third generation of gene editing tool, has been widely used for genome editing in varous cells and organisms. Despite the efficient of gene mutation triggered by guide RNA/ cas9, the specificity and off-target effects can’t be neglected. Recently, a double nicking strategy using catalytic domain mutant Cas9D10Ahas been employed to minimize the off-target effect. Here we developed paired Guide RNAs/nickase All-in-one system in lentivirus backone to efficiently improve the specificity and minimize the off-target effects. CRISPR/Cas9D10Anickase system contains LentiCRISPR Cas9D10A-puro and LentiCRISPR Cas9D10A-GFP, Lenti nickase-guide.
What is the CRISPR-Cas9 system?
Figure 1. Overview of CRISPR-Cas9 system
CRISPR-Cas9 system is a widely used genome editing tool, which derives from the bacteria adaptive immune mechanism. As an engineered version, nowadays CRISPR-Cas9 system contains one enzyme Cas9 and one guide RNA. This guide RNA can direct cas9 to specific genome locus for further digestion. As shown in Figure 1.
So far, CRISPR-Cas9 system has two main applications: Gene knock-out and Gene knock-in. For gene knockout, once DNA double strand breaks are induced, the cells will repair the break through NHEJ DNA repair pathway which will cause tiny deletion or insertion. For gene knock in. with the homolog recombination template, the cells will be inserted one specific foreign sequence in genome.
However, in large genome such as those of mammalian cells, there are potential sites, which contains one or more mismatches with sgRNA that may be tolerated by Cas9, that is so-called potential off-target sites. Indeed, such off-taget effect may limit the application of CRSPR/cas9 system in genetic modification, especially in clinical genetic disease biotherapy.
Subsequently, to conquer the off-target effect, various methodologies, including FokI-Cas9 fusion nucleases, purified Cas9 ribo-nucleoproteins, rationally-engineered Cas9, modified sgRNAs, or paired catalytic mutant Cas9 nickase,have been taken. Here we developed the paired CRISPR/cas9(D10A) system, which significantly reduces the off-target effects and improves the specificity.
Introduction of paired CRISPR/Cas9D10A nickase system
Unlike wild type Cas9 mediated DNA double strands break, Cas9 (D10A) digestion leads to single strand break, so called nick, which can be repaired faithfully. Similarly, to create double strand break using Nickase, we designed the paired CRISPR/Cas9 D10A system, which is competent to minimize the off-target effect. As figure2 illustrated, paired Nickase system usually contains one nickase(Cas9D10Aor Cas9 H840A) and two guide RNAs targeting to adjacent region, specifically to one exon. Here we developed Paired CRISPR/Cas9D10Anickase based gene editing All-in-one system in lentivirus backbone, which is completely applicable to hard transfected cell line and primary cell.
Figure 2. Overview of Paired CRISPR/Cas9D10Anickase system
Plasmids for this protocol
To achieve the higher editing efficiencies and crash the hard transfected cells hurdles, lenti viral vector harboring one cas9 protein and two guide RNA expressing cassettes is generated. In this protocol, three plasmids are involved. lentiCRISPR Cas9D10Apuro and lentiCRISPR Cas9D10AGFP contain cas9 and one Guide RNA expressing cassette. Adding another MCS is for the inserting of the second Guide RNA expressing cassette. Lenti nickase guideis generated for the second guide RNA expressing cassette.
lentiCRISPR Cas9D10AGFP contains EGFP for fluorescent sorting; lentiCRISPR Cas9D10Apuro contains puromycin resistant gene for drug selection.
Figure 3. Map for lentiCRISPR Cas9D10A Puro
Figure 4. Map for lentiCRISPR Cas9D10AGFP
Figure 5. Map for Lenti nickase guide
Design paired Guide RNAs
As following picture illustrated, paired guide RNAs target sense strand and anti-sense strand respectively. We design two guide RNA sited in adjacent region of the same exon in opposition direction. The gap between two guide RNAs can be several base pairs to dozens of base pairs. Make sure that two guide RNAs targeting two strans in opposition direction.
Figure 6. Strategy for designing paired guide RNAs
--------Procedure--------
I. Pick guide RNAs with minimal off-targets potentials
Website for target prediction
http://crispr.mit.edu/. The brief manual is as below (Figure 7.)
1. Input no more than 250 nts’ genomic DNA sequence to the input box, and pick the corresponding species. Then click button ‘submit’.
2. Click button ‘Guides & offtargets’.
3. Choose targets whose scores > 90. The targets can be in both orientations.
Figure 7. Submission of prediction of guide RNA target
II. Construct Cas9 D10A nickase vector carrying paired Guide RNA expressing cassettes
Figure 10. Overview of construction
1. Synthesis target oligos.
Oligo-Forward: CACC-(N)20
Oligo-Reverse: AAAC-(N)20RC
If the first nucleotide of the target sequence is not ‘G’, just add one ‘G’ to the 5’ end. The oligos become
Forward: CACC-G(N)20
Reverse: AAAC-(N)20RC
2. Annealing
On thermal cycler with heat lid.
37 ℃, 30 min
95 ℃, 5 min
Ramp down to 25℃ at 0.1℃/sec. (when step 25℃ is highlighted, click option, set Ramp as 0.1℃/sec)
25 ℃, 5 min
4 ℃, 5 min
Put on ice, or keep in -20℃ freezer for storage.
3. Digest lentiCRISPR D10A Puro (or lentiCRISPRD10A GFP) with Bsm BI and lenti Nickase guide with BbsI
Purify the enzyme digested product with PCR product purification kit, and make sure that the concentration is more than 50 ng/ul.
4. Ligation
Dilute the annealed target oligos 200 times for further ligation
Ligate for more than 2 hours at 16 ℃. (Overnight is better.)
5. Transformation and identification of positive clones
Pick single clones and culture for several hours in 37℃ incubator, and perform PCR to identify the positive clones.
For lentiCRISPR Dual Puro and lentiCRISPR Dual GFP, use Oligo-Forward and CAG-R as primers; for Topo-Dual, use Oligo-Forward and Topo-F as primers.
6. Digest Guide RNA Target2 expressing cassette in the lenti Nickase guide and lentiCRISPR D10A Puro-Target1(or lentiCRISPR D10Al GFP-Target1)
7. Gel extraction and purification
For lenti nickase guide-Target2, cut and purify the smaller DNA band about 360 bps; for the lenti CRSPR D10A pro/GFP-Target1, cut the unique main band about 15,000 bps.
8. Ligation
Ligate overnight at 16 ℃.
9. Transformation and identification of positive clones
Use Oligo-Forward (target1) and CAG-R as primers, and the PCR product is about 600 bp.
10. Plasmid extraction and sequencing validation.
Culture the positive clone in 10 ml Amp+ LB medium, and extract plasmid.
Use CAG-R , Topo-F as sequencing primer.
III. 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 1.5ml conical tube:
For per100mm petri dish:
10 μg Lentivirus backbone plasmid harboring inteterest gene
4 μg pdR8.2. (structural vector)
2 μg pMD2.G (envelope vector)
16 μg in total
3. Add filtered water to a final volume of 878μ1.
4. Add 122μ1 2M CaCl2 and mix.
5. Drop 1000ul of 2xHBS to the abvove mixture slowly. Note: Shaking the tube continuously to avoid generating large particles
6. Sit mixture at room temperature for 2min.
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 M CaCl2
2xHBS: 50mM HEPES
10mM KCl
12mM Dextrose
280mM NaCl
1.5mM Na2HPO4
Resuspend in 900 ml H2O, 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.
III. Knockout in interested cells
Theoretically, any cells transfected with plasmid should be KO cells. However, to get pure KO clones, single clones culture are necessary. Culture of single clones is a little difficult for some cell types. To improve the expansion of single cells, gelatin pre-coating and conditional medium can increase the survival rate of single cells.
1. Plate the cells in 6-well plate one day before, and make sure the cell confluency reach about 70-80%.
2. Transfect the interested cells with validated px459M based plasmid and culture for 24 hours.
3. Treat the transfected cells with 2ug/ml puromycin for 3 or 4 days. Set one mock un-transfected well as control.
Cautions!: before treatment, a pilot experiment is needed to explore the optimized puromycin concentration.
4. Withdraw the puromycin, and culture the cells with 20% FBS medium.
5. Several days later, if independent clones expand, pick the clones and transfer them into 24-well plate for culture. The remained cells keep growing until about 50% cell confluency.
6. When the cells in 24-well plate reach more than 30% confluency, trypsin-digest these wells, take out 80% cells for genotyping, and remain 20% cells for further culture.
7. Quick-spin the cell suspension at 14000rpm for 30s. Remove the supernatant, and lyse the cell pellet with 20-60ul solution A. Heat the lysis at 95 ℃ for 10 min, then add the same volume solution B, vortex for a while, and centrifuge at 13000rpm for 10min. remove the supernatant for PCR template.
8. Do the genotyping, and keep the clones containing knocked-out cells
9. When the clones containing knocked-out cells reach full confluency, digest the cells with trypsin, and count the cell number. Serially dilute the cells in 40 cells/10ml medium by 10-fold.
10. The medium for the step 9 should be the conditional medium. That is, coll ecting medium supernatant of the same cell type (culture time is less than or about 24 hours, avoiding the over-proliferating’s medium supernatant). Add 20% FBS in this supernatant and filter the medium with 0.22 ul filter.
11. Take out 96-well plates, add 100 ul autoclaved 0.1% gelatin solution per well, then incubate in 37 ℃ for at least 30 min.
12. Aspirate the gelatin, and aliquot 100 ul diluted 0.4 cell containing conditional medium per well. Each 8 or 10 wells are added, shake the cell suspension and continue.
13. Add another 100ul conditional medium into the 96-well plate on the fourth or fifth day, and no later than the fifth day.
14. When the cell confluency reach more than 30%, or the clones expand large enough, digest the cells and transfer them into 24-well plate, repeat the step6-8. Keep pure knocked-out clones for further experiment.
15. For the remained cells in step 5. When cell reach 50% confluency, they can also be used for limiting dilution for single clones. Or this mixture can be frozen for later isolation.
VII. Assess off targets effect
Once some clones are identified KO ones. The potential off-target effect should be detected. Website (http://crispr.mit.edu/.) will list all potential off-targets site. Amplify those high-risky off-target site, and examine whether off-target occur through sanger sequency. If any obvious off-targets are not detected, then these clones can be used for further functional experiment.
P.S. Method for quick extraction cell genomic DNA
1.Collect about 2×105 cells, and discard the supernatant.
2.Add 75 μl 25mM NaOH/0.2mM EDTA.(Solution A).
3.Place in thermocycler at 98℃ for 1 hours, then reduce the temperature to 15 ℃
Until ready to proceed to the next step.
4. Add 75 μl of 40mM Tris HCl(pH 5.5).(Solution B)
5. Centrifuge at 4000rpm for 3 minutes.
6. Take an aliquot for PCR (use 2 μl of a 1:100 dilution/reaction).
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